CN1241210A

CN1241210A - Method for generation of primordial germ cell and transgenic animal species

Info

Publication number: CN1241210A
Application number: CN97180552A
Authority: CN
Inventors: J·A·皮德拉希塔; F·W·巴泽尔
Original assignee: Texas A&M University System
Current assignee: Texas A&M University System
Priority date: 1996-10-11
Filing date: 1997-10-10
Publication date: 2000-01-12

Abstract

Disclosed are methods for the isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, methods for transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals. The efficiency at which transgenic animals are generated by the present invention is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.

Description

Produce the method for primordial germ cells and transgenic animal species

Background of invention

1. invention field

The present invention relates generally to the transgenic animal field.In particular, the present invention relates to produce primordial germ cells deutero-clone, transform primordial germ cells and primordial germ cells derived cell system and use these cell transformed and the method for the non-rodent species of clone generation transgenosis.

2. description of Related Art

Have some proterties that needs or feature animal (as put on weight, feeding efficiency, the moiety of meat, milk production or composition and to the resistibility of disease) be that people are needed always.Traditional method for breeding can produce the animal of the proterties that has some needs, but these proterties are usually followed many bad features, and is a method that cost is extremely high and time-consuming.

Exploitation transgenic animal technology has the animal prospect of special requirement proterties long-range for generation.Transgenic animal are to be carried at growth to be introduced the intragentic animal of somatocyte and sexual cell in early days intentionally.Though produced transgenic animal in several different plant species by the whole bag of tricks, also the method for the repeatable large-scale transgene mammal of generation is still deficient easily with rational cost.

At present, the only technology that can be used for producing breeding transgenic livestock is to rely on the pronucleus injection or use virus vector.In these two situations, foreign DNA inserts at random, and this can cause many problems.Matter of utmost importance wherein is to insert inactivation, and this is a kind of inactivation of indispensable gene, and reason is that foreign DNA has destroyed coding or regulated sequence.Another problem is that described transgenosis may not integrated fully, or integrates and but do not express.Further problem is the possibility that exists out of true to regulate owing to position effect.This relates to the instability with gene expression dose and generegulation tolerance range between the difference person of foundation animal of same transgenosis construct generation.Therefore, be not rare to be, produce 10 person of foundation animals and only identify one to guarantee that the mode of keeping transgenic strain expresses described genetically modified animal.

In addition, use prior art, fully deactivation or remove gene in the transgenic animal, and can only add new gene.Consequently, the gene that relates to the undesirable cell process can not be removed, maybe any genetic modification that needs to change existing gene can not be implemented.And, the inefficiency of generation breeding transgenic livestock, one is the efficient unrare (Wall, 1996) of transgenic animal in the offspring of 100 generations.Consequently, produce the cost that transgenic animal followed and to express 250,000 to 500,000 dollars of animals up to every.

By utilizing homologous recombination method (Koller and Smithies, 1992) to overcome these shortcomings, this method instructs described transgenosis to be inserted into certain location.This technology makes can accurately modify the gene of existence, and has overcome the problem of position effect and insertion inactivation.In addition, its make can deactivation special genes and replace another gene with a gene.Regrettably, the efficient of described method is too low so that it can not be directly used in the embryo, and must utilize carrier cell system.The operability of suitable clone will be allowed accurate operation genomic material, produce the Live Animals of carrying those changes subsequently.

Do the ability that (ES) cell has unlimited breeding under the undifferentiated state from the isolating embryo of inner cell matter (ICM) of pre-implantation embryos, and when being injected into the host embryo, can work the healthy tissues of chimeric individuality and the formation of organ.Described ES clone is allowed manipulation in vitro and screening, produces the transgenic animal of carrying those changes subsequently.Cultivating also, the ability of genetic manipulation rear clone sexual cell has made the ES cell become the powerful tool of modifying mouse species gene group.The ES cell of genetic modification and the mosaic that fetal tissues is produced have been used to study vivo gene and have regulated (Stewart etc., 1985) and study the germ cell line transmission (Smithies1991) that is introduced into gene.In addition, by homologous recombination, the ES cell has been used to study the directed modification (Smithies 1991) of gene.

In producing transgenic mice, it is effective that chimeric use has demonstrated.About 70% the mouse blastocyst that rises big is grown the young mouse that survives, wherein 50% be mosaic (Bradley etc., 1984).20% of these chimeric young mouse have the sexual cell chimerism.Utilize this method, the chimerism in might described sexual cell can account for 20-30%.Yet described ES cell method is not successfully applied to the bigger transgenic animal of generation, for example transgenic pig, ox, goat or sheep as yet.The reason that is generalized to than the large mammals failure from the method for mouse may be the difference (Wheeler, 1996) of the etap of described species.

Recently, existing reporting from the behavior of primordial germ cells (PGC) deutero-mouse cell lines is similar to the ES cell and can work to described germ cell line (Labosky etc. 1994).Be called as embryonic genital cell (EG) or PGC derived cell (Labosky etc., 1994; Strelchenko, 1996) these cells make after the mark of undifferentiated state and injection enter host's blastocyst and are different from ES cell (Labosky etc., 1994 aspect the ability of described germ cell line colonyization; Stewart etc., 1994).Therefore, even initial tissue-derived or cell phenotype is different from ICM derived cell system, in case set up, even they inequalityly also have similar character.

Though great majority carry out in mouse ES and genitaloid research, the existing report of the trial of this technology of exploitation in other mammalian species.Have and describe embryo cell line from hamster (Doetschman etc., 1988), mink (Sukoyan etc., 1992,1993), rabbit (Graves and Moreadith, 1993; Giles etc., 1993), pig (Piedrahita etc., 1990; Strojek etc., 1990; Notarianni etc., 1990; Talbot etc., 1998; Wheeler, 1994; Gerfen and Wheeler, 1995; Shim and Anderson, 1995), sheep (Handyside etc., 1987; Piedrahita etc., 1990; Notarianni etc., 1991; Campbell etc., 1995) and ox (Saito etc., 1992; Sims and First, 1998; Stice etc., 1994; Strelchenko, 1996; Stice and Strelchenko, 1996).Though each in these clones some from the described ES cell characteristic of mouse, the sexual cell transmission prerequisite of strain (produce transgenic animal) does not obtain proof as yet.

Producing another problem that transgenic animal followed is DNA Transformed E S or the EG cell that is difficult to having required proterties.These difficulties relate to when going down to posterity repeatedly, and cell can not remain unchanged (not breaking up).These are obviously different with mouse ES cells, and it can repeatedly go down to posterity and not have the obvious change that produces transgenic animal potentiality aspect.So far, in any non-rodents domestic animal species, still there is not the relevant undifferentiated report that is transformed the transgenic cell line aspect that produces embryonic derived or PGC derived cell system.

The mosaic that can participate in enucleation oocyte forms or consideration convey moves growth common by Transformed E S or PGC derived cell system to medical treatment, animal doctor and agriculture field can be very valuable.In medical treatment and veterinary applications, it will allow in dairy products produce biological medicine and oral immunity former, generation can be used as the animal of people tissue donor, but the exploitation accelerated development comprises the animal model of human disease and the exploitation blood substitute of the surrogate therapeutic method of gene therapy.In veterinary applications, it will allow the natural immunity animal of generation to disease specific.At agriculture field, it will allow change dairy products moiety with the increase quality guaranteed period, cheese yield, and make a lactose intolerance dairy products that the physical efficiency safe edible is modified.It also allows the gene alteration that introducing is little, and described change can improve disease resistance, the moiety of growth velocity and meat, the moiety of timber and nutrient efficiency etc.Regrettably, still not having relevant non-rodents ES of conversion or PGC derived cell so far is the description of aspect.

Summary of the invention

By the separation primordial germ cells are provided, cultivate these cells to produce the method for primordial germ cells derived cell system, by the method that transforms described primordial germ cells and described culturing cell system is provided, use these transformants and clone to produce the method for transgenic animal with passing through, the invention solves the problem that produces non-rodents transgenic animal in the prior art.The efficient that the present invention produces transgenic animal is obviously increased, and allows thus in producing the non-rodent species of transgenosis and uses homologous recombination method.

Therefore, the invention provides the genitaloid method of cultivating non-rodent species, be included in feeder cell upper flat plate inoculation primordial germ cells, feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, its substratum contains the Prostatropin of significant quantity.In some aspects, described method can be included in feeder cell upper flat plate inoculation primordial germ cells, and the density of described feeder cell is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, contain the significant quantity Prostatropin in the substratum, continue to be enough to obtain not break up the time span of primordial germ cells colony.

In alternative arrangement, the invention provides the genitaloid method of cultivating non-rodent species, being included in density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶STO feeder cell upper flat plate inoculation between the cells/square cm contains described non-rodent species embryo's primordial germ cells composition, contains the Prostatropin of significant quantity in its substratum.

Also have on the other hand, the invention provides the genitaloid method of non-rodent species of cultivating, be included in the feeder cell upper flat plate and inoculate the embryo's who contains described non-rodent species genitaloid composition, described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, contain the Prostatropin of significant quantity in substratum, described substratum comprises the solubility STEM CELL FACTOR that non-external source adds.

In further content, the invention provides the genitaloid method of non-rodent species of cultivating, be included in the feeder cell upper flat plate and inoculate the genitaloid composition that contains described non-rodent species embryo, described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, contain the Prostatropin of significant quantity in substratum, described substratum comprises the leukaemia inhibitory factor that non-external source adds.

In further content, the invention provides the genitaloid method of non-rodent species of cultivating, be included in the feeder cell upper flat plate and inoculate the embryo's who contains described non-rodent species genitaloid composition, described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, its substratum contains the Prostatropin of significant quantity, and described substratum comprises solubility STEM CELL FACTOR or the leukaemia inhibitory factor that non-external source adds.

In addition, the invention provides and cultivate the genitaloid method of non-rodent species, be included in non-SI/SI ⁴Or SI-m220 feeder cell upper flat plate inoculation contains the embryo's of described non-rodent species genitaloid composition, and described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, in substratum, contain the Prostatropin of significant quantity.

The present invention also provides the genitaloid method of cultivating non-rodent species, is included in non-SI/SI ⁴Or SI-m220 feeder cell upper flat plate inoculation contains the embryo's of described non-rodent species genitaloid composition, and described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, contain the Prostatropin of significant quantity in substratum, described substratum comprises solubility STEM CELL FACTOR or the leukaemia inhibitory factor that non-external source adds.

In certain preferred aspects, contain the embryo of described genitaloid composition separation from non-rodent species, its step comprises: the embryo who collects non-rodent species, shift out embryo's sex-ridge, in the acceptable solution of biology, cultivate described sex-ridge, destroy described sex-ridge, discharge described primordial germ cells thus and collect described primordial germ cells and contain described genitaloid composition to provide.In certain embodiments, described primordial germ cells are collected by centrifuging.

Of the present invention concrete aspect, primordial germ cells comprise at least a first foreign DNA section.The primordial germ cells that contain foreign DNA are called by the primordial germ cells of genetic transformation.In further embodiment, by electroporation, particle bombardment or calcium phosphate precipitation provide external source, selected DNA section for described primordial germ cells.Of the present invention aspect some, provide selected DNA section for containing the primordial germ cells composition, and the primordial germ cells that contain described selected DNA section are selected and can choose wantonly with the primordial germ cells appropriate separation that does not contain the composition of selected DNA section and open.

The density of described feeder cell is crucial to the success of many methods described herein.About 1.5 * 10 ⁵Cells/square cm and about 10 ⁶In the density range between the cells/square cm, actual density can have difference according to practical application.Therefore, aspect some, the density of described feeder cell can be about 1.5 * 10 of the present invention ⁵Cells/square cm and about 5 * 10 ⁵Between the cells/square cm, about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, about 2 * 10 ⁵Cells/square cm and about 9 * 10 ⁵Between the cells/square cm, about 3 * 10 ⁵Cells/square cm and about 8 * 10 ⁵Between the cells/square cm, about 2 * 10 ⁵Cells/square cm and about 5 * 10 ⁵Between the cells/square cm, about 4 * 10 ⁵Cells/square cm and about 7 * 10 ⁵Between the cells/square cm, about 2.5 * 10 ⁵Cells/square cm and about 7.5 * 10 ⁵Between the cells/square cm, about 5 * 10 ⁵Cells/square cm and about 8 * 10 ⁵Between the cells/square cm, about 1.5 * 10 ⁵Cells/square cm and about 3 * 10 ⁵Between the cells/square cm, about 5 * 10 ⁵Cells/square cm and about 6 * 10 ⁵Between the cells/square cm, about 4 * 10 ⁵Cells/square cm and about 6.5 * 10 ⁵Between the cells/square cm, about 5 * 10 ⁵Cells/square cm and about 1 * 10 ⁶Between the cells/square cm, about 8 * 10 ⁵Cells/square cm and about 9 * 10 ⁵Between the cells/square cm, about 2.5 * 10 ⁵Cells/square cm and about 5 * 10 ⁵Between the cells/square cm, or any combination of the interior density of this scope.

Therefore, with regard to specific embodiments, the density of feeder cell original seed can be about 1.5 * 10 ⁵Cells/square cm, 2 * 10 ⁵Cells/square cm, 2.5 * 10 ⁵Cells/square cm, 3 * 10 ⁵Cells/square cm, 4 * 10 ⁵Cells/square cm, 5 * 10 ⁵Cells/square cm, 6 * 10 ⁵Cells/square cm, 7 * 10 ⁵Cells/square cm, 7.5 * 10 ⁵Cells/square cm, 8 * 10 ⁵Cells/square cm, 9 * 10 ⁵Cells/square cm, or 1 * 10 ⁶Cells/square cm.The another way of representing described feeder cell density is by calculating the employed feeder cell number in per 35 millimeters holes.Therefore, the feeder cell density between per 35 millimeters Kong Zaiyue 100 ten thousand and about 900 ten thousand is preferably used in the present invention.Therefore, feeder cell density can be per 35 millimeters holes about 100 ten thousand, about 1.5 hundred ten thousand, about 200 ten thousand, about 300 ten thousand, about 400 ten thousand, about 500 ten thousand, about 600 ten thousand, about 700 ten thousand, about 7.5 hundred ten thousand, about 800 ten thousand, about 8.5 hundred ten thousand, about 900 ten thousand, and per 35 millimeters holes about about 300 ten thousand are particularly preferred in some aspects.

Make and contain described genitaloid separated composition and be grown on one deck feeder cell.Described feeder cell provide microenvironment for genitaloid growth.Described feeder cell provide somatomedin and extracellular matrix are provided for the primordial germ cells of growth.Of the present invention aspect some, described feeder cell system can be by genetically engineered to express selected somatomedin.Therefore, in certain embodiments of the invention, described feeder cell can comprise at least a first exogenous DNA array.The feeder cell typical types that the present invention preferably uses is an embryo cell line, and Tathagata is from the embryo fibroblast of the animal species of selecting (as mouse, pig or ox).Aspect some, described feeder cell can be mouse SI/SI of the present invention ⁴Cell.In other embodiments, described feeder cell can be STO cell (mouse embryo fibroblasts), and other concrete aspect, feeder cell can be SI ⁴-m220 cell.The blended cell culture also can be supposed to be used as the feeder cell of some aspect of the present invention.Therefore, in further content of the present invention, described feeder cell comprise at least a first kind of cell type and at least a second kind of different cell type.In some aspects, described feeder cell are mixtures of STO and pig embryo fibroblast.

Preferably passing through X ray or using the described feeder cell of ametycin deactivation with preceding.In a preferred embodiment of the invention, described feeder cell cobalt ray or the deactivation of caesium ray.

The present invention also is provided at the separated primordial germ cells of cultivation in the suitable substratum.As discussed above, described feeder cell provide somatomedin for the primordial germ cells of growing, yet the amount of the exogenous growth factors that is provided can be different and different with the preparation method of feeder cell.Therefore, aspect some, the somatomedin that external source adds can be added in the fill-in of endogenous use of the present invention.

To the crucial somatomedin of the genitaloid growth of the present invention is Prostatropin.The situation of each somatomedin can be used to the Prostatropin from extensive Mammals source as described herein, includes but not limited to pig, ox, sheep, billy goat, horse, mouse or people.Aspect concrete, rh-bFGF is preferred.In some aspects, somatomedin as Prostatropin, is from the species same with described primordial germ cells, or in others, from the species different with described primordial germ cells.

In preferred embodiments, the substratum of described culture can comprise the rh-bFGF of concentration between about 5 nanograms/milliliter and about 100 mcg/ml.In a more preferred embodiment, described substratum comprises the rh-bFGF of about 40 nanograms/milliliter concentration.Yet, will be understood that described concentration range can be between about 5 nanograms/milliliter and about 10 mcg/ml, or between about 10 nanograms/milliliter and about 100 mcg/ml.Equally, described scope can be between about 10 nanograms/milliliter and about 50 mcg/ml, between about 10 nanograms/milliliter and about 1 mcg/ml or between about 20 nanograms/milliliter and about 250 nanograms/milliliter.

Will also be understood that 5 nanograms/milliliter comprise about 6 nanograms/milliliter, about 7 nanograms/milliliter, about 8 nanograms/milliliter etc., and about 100 mcg/ml comprise about 99 mcg/ml, about 98 mcg/ml, about 97 mcg/ml etc.In addition, the comparable value that provides of the value of described scope low side is low, for example about 4 nanograms/milliliter, and about 3 nanograms/milliliter, and still within the scope of the present invention.Equally, the high-end value of described scope is incited somebody to action 101 mcg/ml according to appointment, and the value of 102 mcg/ml is included in the scope of the present invention.By detecting the influence of the colony growth that different concns and test derive to primordial germ cells, those skilled in the art can be optimized and need not too much test following these or any other medium component concentration.

Aspect some, except Prostatropin, can use other member of fibroblast growth family of the present invention.These members include but not limited to FGF-1 (acid fibroblast growth factor), FGF-3 (int-2), FGF-4 (hst/K-FGF), FGF-5, FGF-6 and FGF-7.

Other somatomedin can join in the described substratum in a kind of significant quantity of undifferentiated state according to promoting primordial germ cells growth characteristics or help to keep primordial germ cells.Therefore, in specific embodiments, described substratum also can comprise the leukaemia inhibitory factor of significant quantity.In some aspects, described substratum comprises the leukaemia inhibitory factor of concentration between about 5 nanograms/milliliter and about 100 mcg/ml.In a more preferred embodiment, described substratum comprises the leukaemia inhibitory factor of concentration between about 10 nanograms/milliliter and about 10 mcg/ml.In a more preferred embodiment, described substratum comprises the leukaemia inhibitory factor of concentration between about 15 nanograms/milliliter and about 1 mcg/ml.In particularly preferred embodiments, described substratum comprises the leukaemia inhibitory factor of about 20 nanograms/milliliter concentration.Yet, will be understood that described concentration range can be between about 5 nanograms/milliliter and about 10 mcg/ml or between about 10 nanograms/milliliter and about 100 mcg/ml.Equally, described scope can be between about 10 nanograms/milliliter and about 50 mcg/ml, between about 10 nanograms/milliliter and about 1 mcg/ml or between about 20 nanograms/milliliter and about 250 nanograms/milliliter.

Should also be understood that about 5 nanograms/milliliter comprise about 6 nanograms/milliliter, about 7 nanograms/milliliter, about 8 nanograms/milliliter etc., and about 100 mcg/ml comprise about 99 mcg/ml, about 98 mcg/ml, about 97 mcg/ml etc.In addition, the value that is provided can be provided the value of described scope low side, and for example about 4 nanograms/milliliter or about 3 nanograms/milliliter are also still within the scope of the invention.Equally, the high-end value of described scope comprise 101 mcg/ml according to appointment or about 102 mcg/ml value within the scope of the present invention.

In other embodiments, described substratum also can comprise the uterus ferritin (uteroferrin) of significant quantity.In certain embodiments, described substratum comprises the uterus ferritin of concentration between about 1 nanograms/milliliter and about 100 mcg/ml.In certain embodiments, described substratum comprises between about 10 nanograms/milliliter and about 5 mcg/ml or the uterus ferritin of concentration between about 20 nanograms/milliliter and about 500 nanograms/milliliter.Aspect some, described substratum comprises the uterus ferritin of about 40 nanograms/milliliter concentration of the present invention.In other embodiments, described substratum also can comprise the solubility STEM CELL FACTOR of significant quantity.Specifically, described substratum comprises the solubility STEM CELL FACTOR of concentration between about 1 nanograms/milliliter and about 100 micrograms.In other embodiments, described substratum comprises the solubility STEM CELL FACTOR of concentration between about 10 nanograms/milliliter and about 5 mcg/ml.Also have in the preferred embodiment, described substratum comprises the solubility STEM CELL FACTOR of concentration between about 20 nanograms/milliliter and about 250 mcg/ml.In typical embodiments, described substratum comprises the solubility STEM CELL FACTOR of about 40 nanograms/milliliter concentration.Yet the concentration range that will be understood that these factors can be between about 1 nanograms/milliliter and about 10 mcg/ml, or between about 10 nanograms/milliliter and about 100 mcg/ml.Equally, described scope can be between 10 nanograms/milliliter and about 50 mcg/ml, between about 10 nanograms/milliliter and about 1 mcg/ml or between about 20 nanograms/milliliter and about 250 nanograms/milliliter.

Those skilled in the art will be understood that about 1 nanograms/milliliter comprises about 2 nanograms/milliliter, about 3 nanograms/milliliter, and about 4 nanograms/milliliter etc., and about 100 mcg/ml comprise about 99 mcg/ml, about 98 mcg/ml, about 97 mcg/ml etc.In addition, the value that is provided can be provided the value of described scope low side, and for example about 0.8 nanograms/milliliter, about 0.5 nanograms/milliliter are also still within the scope of the present invention.High-end 101 mcg/ml according to appointment of same described scope, or the value of about 102 mcg/ml is included in the scope of the present invention.

In further embodiment, described substratum also can comprise the alpha2-macroglobulin of significant quantity.Specifically, described substratum comprises the alpha2-macroglobulin of concentration between about 10 nanograms/milliliter and about 10 mcg/ml.In other embodiments, described substratum comprises the solubility STEM CELL FACTOR of concentration between about 50 nanograms/milliliter and about 5 mcg/ml.Also have in the preferred embodiment, described substratum comprises the solubility STEM CELL FACTOR of concentration between about 100 nanograms/milliliter and about 2.5 mcg/ml.In typical embodiments, described substratum comprises the solubility STEM CELL FACTOR of about 1 mcg/ml concentration.Yet, will be understood that the concentration range of these factors can be between about 10 nanograms/milliliter and about 2.5 mcg/ml, or between about 100 nanograms/milliliter and about 10 mcg/ml.Equally, described scope can be between about 100 nanograms/milliliter and about 5 mcg/ml, between about 250 nanograms/milliliter and about 2.5 mcg/ml or between about 500 nanograms/milliliter and about 1 mcg/ml.

Those skilled in the art will be understood that about 10 nanograms/milliliter comprise about 11 nanograms/milliliter, about 12 nanograms/milliliter, about 13 nanograms/milliliter, about 14 nanograms/milliliter etc., and about 10 mcg/ml also comprise about 9 mcg/ml, about 8 mcg/ml, about 7 mcg/ml etc.In addition, the value that is provided can be provided the value of described scope low side, and for example about 8 nanograms/milliliter, about 5 nanograms/milliliter are also still within the scope of the present invention.Same described scope is high-end will to be worth 11 mcg/ml according to appointment, or about 12 mcg/ml are included in the scope of the present invention.

The invention provides some embodiment, wherein said substratum also can comprise the amino acid to the nonessential significant quantity of described concrete non-rodent.In further embodiment, described substratum comprise concentration between about 10nM and the about 250nM to the nonessential amino acid of concrete non-rodent.In addition, described substratum comprise concentration between about 50nM and the about 150nM to the nonessential amino acid of concrete non-rodent.Also have in other embodiment, described substratum comprise about 100nM concentration to the nonessential amino acid of concrete non-rodent.Yet, will be understood that described concentration range can be between about 10nM and about 100nM, or between about 20nM and about 250nM.Equally, described scope can be between about 20nM and about 150nM, between about 50nM and about 125nM, or between about 75nM and about 110nM.

Should also be understood that 10nM comprises about 11nM, about 12nM, about 13nM etc., and about 250nM comprises about 249nM, about 248nM, about 247nM etc.In addition, the value that is provided can be provided the value of described scope low side, and for example about 9nM, about 8nM are also still within the scope of the present invention.Equally, the value of the high-end 251nM according to appointment of described scope or about 252nM is included in the scope of the present invention.

In a preferred embodiment of the invention, described substratum also can comprise the L-glutaminate of significant quantity.Specifically, described substratum comprises the L-glutaminate of concentration between about 0.1mM and the about 50mM.In more preferred, described substratum comprises the L-glutaminate of concentration between about 1mM and the about 20mM.Also have in the preferred embodiment, described substratum comprises the L-glutaminate of about 2nM concentration.Yet, will be understood that described concentration range can be between about 0.1mM and about 10mM, or between about 0.5mM and about 50mM.Equally, described scope can be between about 0.7mM and about 10mM, between about 1mM and the about 5mM or between about 1.5mM and about 2.5mM.

Should also be understood that about 0.1mM comprises about 0.2mM, about 0.3mM, about 0.4mM etc., and about 50mM comprises about 49mM, about 48mM, about 47mM etc.In addition, the value that is provided can be provided the value of described scope low side, and for example about 0.09mM, about 0.08mM are also still within the scope of the present invention.Equally, 51mM or about 52mM belong in the scope of the invention the high-end value that comprises of described scope according to appointment.

In other preferred embodiment of the present invention, described substratum also can comprise the β mercaptoethanol of significant quantity.In some aspects, described substratum comprises the β mercaptoethanol of concentration between about 1 μ M and the about 1mM.In further embodiment, described substratum comprises the β mercaptoethanol of concentration between about 25 μ M and the about 250 μ M.In typical embodiments, described substratum comprises the β mercaptoethanol of about 100 μ M concentration.Yet, will be understood that described concentration range can be between about 1 μ M and about 500 μ M, or between about 5 μ M peace treaties or 1mM.Equally, described scope can be between about 20 μ M and about 250 μ M, between about 50 μ M and about 125 μ M, or between about 75 μ M and about 110 μ M.

Should also be understood that about 1 μ M comprises about 0.9 μ M, about 0.8 μ M, about 0.7 μ M etc., and about 1mM comprises about 2mM, about 3mM, about 4mM etc.In addition, the value that is provided can be provided the value of described scope low side, for example about 0.9 μ M, or about 0.8 μ M is also still within the scope of the present invention.Equally, 2mM or about 3mM belong in the scope of the invention the high-end value that comprises of described scope according to appointment.As discussed above, by detecting different concns and the test influence to primordial germ cells deutero-colony, those skilled in the art can be optimized and need not too much experiment this or other medium component concentration.

In certain embodiments, described substratum also can comprise the improved Eagle substratum of Dulbecco of significant quantity.The improved Eagle substratum of Dulbecco can be low improved Eagle substratum of sodium Dulbecco or the improved Eagle substratum of high sodium Dulbecco.In typical embodiment, described substratum comprises the improved Eagle substratum of Dulbecco of about 50% volume/volume.In other embodiments, described substratum also can comprise Ham F10 substratum.In a more preferred embodiment, described substratum comprises the Ham F10 substratum of about 50% volume/volume.In typical embodiments of the present invention, described substratum comprises the Ham F10 substratum of the improved Eagle substratum of about 50% volume/volume Dulbecco and about 50% volume/volume.Obviously, improved Eagle substratum of Dulbecco or Ham F10 substratum can be about 40% volume/volume, about 30% volume/volume etc.In addition, about 50% comprise about 49%, about 48% grade and about 51%, about 52% and about 53% and still keep within the scope of the present invention.

The substratum that contains different growth factor combinations also is supposed to use in the present invention.Therefore, of the present invention aspect some, described substratum comprises the Prostatropin of significant quantity and the uterus ferritin of at least a significant quantity, alpha2-macroglobulin, leukaemia inhibitory factor, solubility STEM CELL FACTOR, to the nonessential amino acid of described non-rodent, L-glutaminate, beta-mercaptoethanol, improved Eagle substratum of Dulbecco or Ham F10 substratum.In further content, described substratum comprises the Prostatropin of significant quantity and in conjunction with at least two kinds of uterus ferritins of significant quantity, alpha2-macroglobulin, leukaemia inhibitory factor, the solubility STEM CELL FACTOR is to the nonessential amino acid of described non-rodent, L-glutaminate, beta-mercaptoethanol, improved Eagle substratum of Dulbecco or Ham F10 substratum.

In a preferred embodiment of the invention, described substratum comprises the Prostatropin of significant quantity and at least three kinds the uterus ferritin in conjunction with significant quantity, alpha2-macroglobulin, leukaemia inhibitory factor, the solubility STEM CELL FACTOR is to the nonessential amino acid of described non-rodent, L-glutaminate, beta-mercaptoethanol, improved Eagle substratum of Dulbecco or HamF10 substratum.In further content of the present invention, described substratum comprises the Prostatropin of significant quantity and in conjunction with uterus ferritin, alpha2-macroglobulin and the leukaemia inhibitory factor of significant quantity.In specific embodiments, described substratum comprises the Prostatropin of concentration between about 5 nanograms/milliliter and about 100 mcg/ml, the uterus ferritin of concentration between about 1 nanograms/milliliter and about 100 mcg/ml, the leukaemia inhibitory factor of concentration between the alpha2-macroglobulin of concentration and about 5 nanograms/milliliter and about 100 mcg/ml between about 10 nanograms/milliliter and about 10 mcg/ml.

In certain embodiments of the invention, described substratum comprises the Prostatropin between about 5 nanograms/milliliter and about 100 mcg/ml, uterus ferritin between about 1 nanograms/milliliter and about 100 mcg/ml, alpha2-macroglobulin between about 10 nanograms/milliliter and about 10 mcg/ml, leukaemia inhibitory factor between about 5 nanograms/milliliter and about 100 mcg/ml, solubility STEM CELL FACTOR between about 1 nanograms/milliliter and about 100 mcg/ml, non-essential amino acid between about 10nM and the about 250nM, L-glutaminate between about 0.1mM and the about 50mM, β mercaptoethanol between about 1 μ M and the about 1mM, the Ham F10 substratum of the improved Eagle substratum of the Dulbecco of about 50% volume/volume and about 50% volume/volume.

In the typical embodiments of the present invention, described substratum comprises the Prostatropin of about 40 nanograms/milliliter, the uterus ferritin of about 40 nanograms/milliliter, the alpha2-macroglobulin of about 1 mcg/ml, the leukaemia inhibitory factor of about 20 nanograms/milliliter, the solubility STEM CELL FACTOR of about 40 nanograms/milliliter, the non-essential amino acid of about 100nM, the L-glutaminate of about 2mM, the β mercaptoethanol of about 0.1mM, the Ham F10 substratum of the improved Eagle substratum of the Dulbecco of about 50% volume/volume and about 50% volume/volume.

The present invention also provides the primordial germ cells of keeping plating in undifferentiated state about 2 generations, about 3 generations, about 4 generations, about 5 generations, about 6 generations, about 7 generations, about 8 generations, about 9 generations, about 10 generations, about 11 generations, about 12 generations, the method in about 13 generations or about 14 generations.In other embodiments of the present invention, the primordial germ cells of plating are maintained at undifferentiated state about 20 generations, about 30 generations, about 50 generations or about 100 generations.

Term used herein " non-rodent " will be understood to include the whole vertebratess except that the rodents and the mankind.In certain embodiments of the invention, non-rodent species are oxen.In other embodiments, described non-rodent kind is a sheep.Also have in other the embodiment, described non-rodent species are pigs.Also have in other embodiment, described non-rodent species are billy goats.Other non-rodent that expectation is used in the present invention includes but not limited to horse, buffalo and rabbit.

The invention provides the primordial germ cells of non-rodent species, its compound method can comprise that plating contains the primordial germ cells composition 1.5 * 10 ⁵Cells/square cm and about 10 ⁶On the feeder cell original seed of density, in substratum, comprise the Prostatropin of significant quantity between the cell square centimeter.In specific embodiments, the invention provides the primordial germ cells colony of non-rodent species, contain the primordial germ cells composition 1.5 * 10 by comprising plating ⁵Cells/square cm and about 10 ⁶Process between the cell square centimeter on the feeder cell original seed of density can prepare described primordial germ cells colony, and its substratum comprises the Prostatropin of significant quantity, continues to be enough to obtain the time span of primordial germ cells colony.Aspect preferred, described primordial germ cells colony is a undifferentiated state.

The invention provides the method for the primordial germ cells derived cell system of the non-rodent of preparation in addition, described method can comprise that plating contains genitaloid composition to about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶On the feeder cell original seed of density, in substratum, contain the Prostatropin of significant quantity between the cells/square cm, and the primordial germ cells of cultivating described plating continue to be enough to provide the time span of primordial germ cells derived cell system.

Therefore, the invention provides the primordial germ cells derived cell system of non-rodent species, contain the primordial germ cells composition about 1.5 * 10 by comprising plating ⁵Cells/square cm and about 10 ⁶Process between the cell square centimeter on the feeder cell original seed of density can prepare described primordial germ cells derived cell, the Prostatropin that in substratum, comprises significant quantity, and the primordial germ cells of culture plate inoculation, continue to be enough to obtain the time span that the primordial germ cells derived cell is.

In addition, the invention provides the genitaloid method that preparation contains the non-rodent species of selected DNA section, described method can comprise introduce selected DNA section in the genitaloid composition that contains non-rodent species obtaining to contain candidate's primordial germ cells of selected DNA section, and about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶The inoculation of the feeder cell upper flat plate of density contains candidate's primordial germ cells of the non-rodent of selected DNA section between the cells/square cm, the Prostatropin that comprises significant quantity in substratum is with the primordial germ cells of the non-rodent species that obtain to contain selected DNA section.

Specifically, the step that described method can comprise is: selected DNA section is incorporated into contains in the genitaloid composition of non-rodent species to obtain to contain candidate's primordial germ cells of selected DNA section, screening has described candidate's primordial germ cells of non-rodent species of selected DNA section, and is about 1.5 * 10 in density ⁵Cells/square cm and about 10 ⁶Feeder cell upper flat plate inoculation between the cells/square cm contains candidate's primordial germ cells of the non-rodent of selected DNA section, the Prostatropin that comprises significant quantity in its substratum continues to be enough to obtain to contain the time span of the non-rodent species primordial germ cells colony of selected DNA section.

In further content of the present invention, the step that described method comprises is: in density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Feeder cell upper flat plate inoculation between the cells/square cm comprises genitaloid composition, the Prostatropin that comprises significant quantity in its substratum, continue to be enough to obtain the time span of the first-generation at least, selected DNA section is incorporated into contains in the genitaloid composition of non-rodent species obtaining to contain candidate's primordial germ cells of selected DNA section, and be about 1.5 * 10 in density ⁵Cells/square cm and about 10 ⁶Feeder cell upper flat plate inoculation between the cells/square cm contains candidate's primordial germ cells of the non-rodent of selected DNA section, the Prostatropin that comprises significant quantity in its substratum is with the primordial germ cells of the non-rodent species that obtain to contain selected DNA section.

In concrete grammar of the present invention, the primordial germ cells that contain the non-rodent species of selected DNA section are cultivated about 2 to 14 generations.In other preferred method, the primordial germ cells that contain the non-rodent species of selected DNA section are cultivated about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 or about 13 generations.

In typical embodiments of the present invention, introduce selected DNA section in described primordial germ cells by electroporation.In other method, by the particle bombardment method, calcium phosphate conversion method or introduce selected DNA section in described primordial germ cells by viral conversion method.

In certain embodiments, selected DNA section can comprise selected proteinic at least the first coding region of coding, and express in one or more described primordial germ cells wherein said coding region.In further embodiment, selected disease resistance, meat moiety, the body weight of increase, the moiety of fur or the dairy component protein of first coding region coding.In other embodiments, the selected labelled protein of first coding region coding.In typical embodiments, the first coding region encoding green fluorescent protein matter, this protein has been adapted to increase in described non-rodent species and has expressed.A kind of protein uses the codon that preferably uses by its encoding sequence of change but " is adapted to increase and expresses " in non-rodent kind in the non-rodent species that specifically need to use.Also has in other the embodiment first coding region coding neomycin resistance protein.In further embodiment, first coding region coding GP63, myelin basic protein matter, hCD59, factors IX, alpha antitrypsin, alpha-casein, a kind of interleukin-or Bc1-2.

In typical embodiments of the present invention, selected DNA section also can comprise a kind of selected proteinic second coding region of coding.In specific embodiments of the present invention, a kind of selected non-marked protein of the first coding region codified is a kind of selected labelled protein and encode in second coding region.

Express in the embodiment of selected DNA section at needs, described DNA section effectively is placed under the control of a promotor, (but being not limited to) that exemplifies promotor of expressible dna section in described primordial germ cells has the CMV promotor, Oct-4 promotor or pgk promotor.In other embodiments of the present invention, selected DNA section is oppositely effectively to be placed on described promotor control down, and wherein said promotor instructs the expression of antisense product.

Also have in other embodiment of the present invention, described DNA section comprises the DNA zone that two of described coding region flank are selected, instructs described coding region homologous recombination thus in the genomic dna of non-rodent species.In a more preferred embodiment, selected DNA zone is corresponding to peculiar sequence in the described non-rodent genomic dna.In typical embodiments, separated DNA zone is corresponding to the Oct-4 gene, or Oct-4 gene flank region.

Also have in other embodiment of the present invention, described DNA section comprises that two selected dna sequence dnas of this DNA section flank and they allow the shearing of carrying out this DNA section under proper condition.In concrete preferred embodiment, described dna sequence dna is the loxP site.

In some preferred method of the present invention, described non-rodent is an ox, sheep, pig, billy goat or horse.In other preferred method, described non-rodent is buffalo or rabbit.

Therefore, the invention provides the primordial germ cells of the non-rodent species that contain selected DNA section, prepare described primordial germ cells by the process that comprises the steps: introducing selected DNA section is about 1.5 * 10 with candidate's primordial germ cells of the non-rodent species that obtain to contain selected DNA section with in density in the separated genitaloid composition that contains non-rodent species ⁵Cells/square cm and about 10 ⁶Feeder cell upper flat plate inoculation between the square centimeter contains candidate's primordial germ cells of the described non-rodent of selected DNA section, the Prostatropin that comprises significant quantity in its substratum is with the primordial germ cells of the non-rodent that obtains to contain selected DNA section.

The present invention also provides the primordial germ cells of the non-rodent that contains selected DNA section, and described primordial germ cells can prepare by the process that comprises the steps: in density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Feeder cell upper flat plate inoculation between the square centimeter contains genitaloid composition, the Prostatropin that in its substratum, comprises significant quantity, selected DNA section is incorporated into contains from the isolating genitaloid composition of non-rodent species with candidate's primordial germ cells of the non-rodent that obtains to contain selected DNA section and be about 1.5 * 10 in density ⁵Cells/square cm and about 10 ⁶Feeder cell upper flat plate between the square centimeter is inoculated described candidate's primordial germ cells, comprises the Prostatropin of significant quantity in its substratum, contains the primordial germ cells of the non-rodent of selected DNA section with acquisition.

In further content of the present invention, the primordial germ cells of the non-rodent species that contain selected DNA section are provided, described primordial germ cells can prepare by the process that comprises following steps: in density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Feeder cell upper flat plate inoculation between the square centimeter contains genitaloid composition, the Prostatropin that in its substratum, comprises significant quantity, introduce selected DNA section to containing from the isolating genitaloid composition of non-rodent species, with candidate's primordial germ cells of the non-rodent that obtains to contain selected DNA section with in density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Feeder cell upper flat plate between the square centimeter is inoculated described candidate and is transformed primordial germ cells, the Prostatropin that in its substratum, comprises significant quantity, and screening has candidate's primordial germ cells of non-rodent species of selected DNA section, with the primordial germ cells of the non-rodent that obtains to contain selected DNA section.

The present invention also provides the method that produces the non-rodent of transgenosis, comprise and introduce selected DNA section in the genitaloid composition that contains described non-rodent, to obtain to contain candidate's primordial germ cells of selected DNA section, inoculate the described candidate's primordial germ cells that contain selected DNA section at the feeder cell upper flat plate, described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, the Prostatropin that in its substratum, contains significant quantity, the described primordial germ cells that contain the described non-rodent of selected DNA section with acquisition, produce the non-rodent of transgenosis with the described primordial germ cells from the non-rodent that contains described selected DNA section, wherein said selected DNA section exists in described non-rodent somatocyte and sexual cell and expresses.

In addition, the invention provides the method that produces transgenic pig, comprise and introduce selected DNA section to containing in the genitaloid composition of pig to obtain to contain the candidate pig primordial germ cells of described selected DNA section, inoculate the described candidate pig primordial germ cells that contain described selected DNA section at the feeder cell upper flat plate, described feeder cell density is about 2 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, the Prostatropin that in its substratum, contains significant quantity, the pig primordial germ cells that contain the described non-rodent of described selected DNA section with acquisition, with produce transgenic pig from the primordial germ cells that contain described selected DNA section, wherein said selected DNA section exists in the somatocyte of described transgenic pig and sexual cell and expresses.

The present invention further provides the method that produces the non-rodent of transgenosis, be included in the inoculation of feeder cell upper flat plate and contain genitaloid composition, described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, the Prostatropin that in its substratum, contains significant quantity, introduce selected DNA section to the genitaloid described composition that contains described non-rodent, to obtain to contain candidate's primordial germ cells of described selected DNA section, inoculate the described candidate's primordial germ cells that contain described selected DNA section at the feeder cell upper flat plate, described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, the Prostatropin that in its substratum, contains significant quantity, the described primordial germ cells that contain the described non-rodent of described selected DNA section with acquisition, produce the non-rodent of transgenosis with the described primordial germ cells from the non-rodent that contains described selected DNA section, wherein said selected DNA section exists in the somatocyte of described non-rodent and sexual cell and expresses.

In addition, the invention provides the method that produces the non-rodent of transgenosis, comprise introduce selected DNA section in the genitaloid composition that contains described non-rodent to obtain to contain candidate's primordial germ cells of described selected DNA section, inoculate the described candidate's primordial germ cells that contain selected DNA section at the feeder cell upper flat plate, described feeder cell density is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, the Prostatropin that in its substratum, contains significant quantity, screen the genitaloid described selected DNA section of described candidate, the described primordial germ cells that contain the described non-rodent of described selected DNA section with acquisition, produce the non-rodent of transgenosis with the described primordial germ cells from the non-rodent that contains described selected DNA section, wherein said selected DNA section exists in the somatocyte of described non-rodent and sexual cell and expresses.

In certain embodiments, comprising genitaloid composition, to contain from the primordial germ cells derived cell be cultured cells.

In specific embodiments of the present invention, enter the non-rodent of method generation transgenosis of the blastocyst that comes by described non-rodent species by a kind of primordial germ cells of injecting the non-rodent species that contain described selected DNA section that comprise.In some aspects, the method that produces the non-rodent of transgenosis comprises that the primordial germ cells of injecting the non-rodent that contains selected DNA section enter the blastocyst of non-rodent species, shift the synchronization that described blastocyst enters non-rodent species and accept the non-rodent that female body is become pregnant with generation, and make the non-rodent gestation of becoming pregnant be enough to for some time that the non-rodent of the transgenosis that can survive is grown.In further embodiment, obtain the non-rodent of transgenosis that can survive by birth naturally, and in other embodiments, be shifted out from the non-rodent of the transgenosis of the survival of accepting female body by operation and obtain the non-rodent of transgenosis alive.

In others of the present invention, the method for the non-rodent of generation transgenosis comprises from the primordial germ cells separating nucleus of the non-rodent that contains selected DNA section and injects the enucleation oocyte that described nucleus enters described non-rodent.In specific embodiments, the method of the non-rodent of generation transgenosis comprises from the primordial germ cells of the non-rodent that contains selected DNA section isolates nucleus, and inject described nucleus and enter enucleation oocyte from described non-rodent species, shift described ovum and accept the non-rodent of becoming pregnant with generation in the female body to the synchronization of described non-rodent species, and for some time that makes described non-rodent gestation of becoming pregnant be enough to make the non-rodent of transgenosis of survival to grow.

Also have in other embodiment of the present invention, the method that produces the non-rodent of transgenosis comprises and flocking together containing the primordial germ cells of non-rodent species of selected DNA section and the body early embryo of described non-rodent species.In some aspects, the method that produces the non-rodent of transgenosis comprises and gathering together containing the primordial germ cells of non-rodent of selected DNA section and the body early embryo of described non-rodent, shift described embryo and accept the non-rodent of becoming pregnant with generation in the female body, and the described non-rodent gestation of becoming pregnant is proceeded to be enough to for some time that the non-rodent of survival transgenosis is grown to the synchronization of described non-rodent species.

The present invention also provides the non-rodent of a kind of transgenosis of preparation by the following method, the step that this method comprises has: introduce selected DNA section and enter the genitaloid composition that contains non-rodent contains described DNA section with acquisition candidate's primordial germ cells, inoculate the candidate's primordial germ cells that contain selected DNA section at the feeder cell upper flat plate, the density of described feeder cell is about 1.5 * 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, the Prostatropin that comprises significant quantity in its substratum, the primordial germ cells that contain the non-rodent species of selected DNA section with acquisition, with produce the non-rodent of transgenosis by the primordial germ cells of the non-rodent that contains selected DNA section, wherein selected DNA section exists in the somatocyte of described non-rodent and sexual cell and expresses.Of the present invention concrete aspect, described transgenic animal are milk cows, sheep, pig, buffalo, horse, rabbit or goat.

The present invention also provides the primordial germ cells that contain non-rodent species, and being enough to reach density is about 1.5 * 10 ⁵With 10 ⁶The feeder cell of feeder cell/square centimeter and promote the growth of described primordial germ cells and the continuous composition of the Prostatropin of the significant quantity of breeding.

Aspect some, described primordial germ cells comprise at least one first foreign DNA section of the present invention.In others, described feeder cell are STO cells.In specific embodiments of the present invention, described composition can further comprise one or more uterus ferritins that promote described primordial germ cells growth and constantly breed significant quantity, alpha2-macroglobulin, leukaemia inhibitory factor, the solubility STEM CELL FACTOR is to the nonessential amino acid of non-rodent species of desired use, L-glutaminate, beta-mercaptoethanol, improved Eagle substratum of Dulbecco and/or Ham F10 substratum.

In further content of the present invention, described primordial germ cells are oxen, sheep, pig, billy goat, horse, the primordial germ cells of buffalo or rabbit.In preferred embodiments, described primordial germ cells are pig primordial germ cells.

The present invention also provide any in preparation primordial germ cells derived cell system the purposes of disclosed composition.Therefore, this composition is supposed to be used to prepare the primordial germ cells derived cell and is.In addition, the invention provides any disclosed purposes that contains the composition of foreign DNA section in the non-rodent of preparation transgenosis.Therefore, the composition that the contains foreign DNA section of the present invention non-rodent of transgenosis that is supposed to be used to prepare.

The present invention also provides all ingredients box that uses in the practice of some open and claimed method of this paper.The invention provides the purposes that comprises genitaloid any open composition in the preparation test kit.Therefore, any described primordial germ cells composition test kit that is supposed to be used to prepare.In particular content, described test kit can comprise the primordial germ cells of (in the appropriate containers mode) non-rodent, is enough to reach about 1.5 * 10 ⁵With about 10 ⁶The feeder cell of the density between feeder cell/square centimeter and promote the growth of described primordial germ cells and the continuous Prostatropin of breeding significant quantity.

The accompanying drawing summary

Following accompanying drawing constitutes the application's part and is included with further proof some aspect of the present invention.The present invention can be sharpened understanding with reference to one of these accompanying drawings or several by the detailed description in conjunction with this paper specific embodiments.

Fig. 1 introduces the WAP locus employed tactful diagram of loxP site to mouse ES cells.Whole four exons all are shown, although the size of exon and relative distance each other do not define.At first, by the WAP allelotrope that leads of the neo-TK box homologous recombination of loxP flank with the exon 4 that is positioned at the guiding construction.The ES cell that is directed to is contacted with the Cre recombinase with the delete flag box stay single loxP site in described genome.These modified ES cells insertion incident by Cre mediation of can being used to will contain the construction specificity of loxP and introduce described WAP locus.During handling, each carries out the isolate of screening process with the required modification of enrichment.

The guiding of mouse WAP gene in Fig. 2 ES cell.With endogenous WAP locus after the pWPNT homologous recombination and the WAP locus that is directed to.The thick line of WAP locus is drawn the homology zone between described native gene seat and the pWPNT oriented carrier.The enzyme site that confirms the use of guiding incident is as follows: E, EcoRI; N, NsiI; S, SphI.Use exon-3 specific probe provides endogenous and is directed to the size that the allelotrope expection is with.The described loxP site usefulness not triangle of filling is represented.

The description of explanatory embodiment

Term used herein " animal " and " non-rodent " comprise the whole vertebrates except rodent and the mankind. Also be included in the animal individual of all stages of development, comprise embryonic stage and tire phase. " transgenic animals " are to contain to carry directly or indirectly to have a mind to genetic manipulation by subcellsular level and accepted any animal of one or more cells of hereditary information. Described genetic manipulation can be carried out to intracellular any method by introducing inhereditary material, includes but not limited to microinjection, recombinant virus infection, particle bombardment or electroporation. Described term is not meaned and is comprised traditional hybridization or in vitro fertilization, but meaning comprises the animal that wherein one or more cells have been accepted recombinant DNA molecules. This molecule can be integrated in the item chromosome, maybe can be the DNA of extrachromosomal replication. Described hereditary information can be external source for the animal species that belongs to acceptor, or is external sources to indivedual acceptors only, maybe can be that described acceptor has and with natural gene varying level arranged, the hereditary information of different time or different local expressions.

The various uses that transgenic animals have widely and constantly enlarge, include but not limited in animal reservoir's milk, produce protein, set up the animal model for research human and animal disease, foundation is to disease and the resistive animal of insect, promote the growth characteristics of animal, improving pork quality constituent and generation can be used as the animal of human blood and tissue donor. No matter the transgenosis of larger animal aspect agricultural and human and animal doctor potential application how, make progress is slowly always. This be since aspect the larger animal transgenics defective of prior art, also because that their generation is followed is expensive.

At present, the whole technology that can be used for domestic animal depend on by pronucleus injection or viral vectors introduces a transgenosis to a chromosomal site at random. In the ideal, in described transgenic animals body, gene expression should with the tissue of simulation with used promoter, be grown the approach of the endogenous expression pattern relevant with temporal and be regulated. Yet this only is desirable. Metallothionein promoter (a kind of derivable liver specificity promoter) causes being similar to the highest transgene level (Palmiter etc. of endogenesis promoter in liver, 1983), but promoter " seepage " also causes ectopic expression in kidney and enteron aisle.

And because the problem that described genetically modified radom insertion is followed, the transgenosis that produces with same construction may be fully different. Another problem that radom insertion is followed is to insert inactivation, and described transgenosis is inserted into the inside of key gene thus, thereby has destroyed key gene. Even this impact is that what to be harmful to is (Schnieke etc., 1983) that cause death to the embryo who is growing.

What bigger dispute was arranged is so-called " position effect ", and wherein transgenosis is inserted into chromosomal zones of different, causes the huge difference of transcriptional level. In the contiguous front and back of dna sequence dna, the inactivation by the localization chromosome structure can make described transgenosis in fact reticent. The actual result of this position effect is the effect owing to adjacent domain, and suitable Gene regulation can be had a strong impact on. Therefore, utilize same construction, can obtain having the transgene expression pattern of simulation endogenesis promoter, even the expression of unusual expression pattern or reduction does not have the transgenic animals (Klintworth, 1990) of expression.

Because the phenotype that transgenic animals produce can not be determined in advance, needs to produce the more than one person of foundation animal to estimate described genetically modified effect. Recently, the factor of existing several cis actings is included in the transgenosis construct to strengthen position independent form expression pattern. These factors comprise: LCRs (locus control region), insulator (insulators) and MAR (matrix attachment regions) (Reitman etc., 1993; McKnight etc., 1992; Krnacik etc., 1995). Although obtained some progress in this field, LCRs and MARs still have to be determined to the universal availability that produces position independent form transgenic animals.

In addition, a kind of incident of transgenosis integration efficiency is particularly when with big domestic animal species. Schindler and Ebert once reported the poor efficiency that produces transgenosis farming animals (pig, sheep, ox, he-goat), and scope is in 0.0% to 4.0% (Ebert and Schindler, 1993). And the mouse transgene efficiency is 10-40% (Palmiter and Brinster, 1986). And in pig, the 60% transgenosis person of foundation expresses the c-ski transgenosis, and only has 38% proof that described phenotype (Ebert and Schindler, 1993) is arranged. The most general worry of the commercial use of transgenics is the poor efficiency that produces transgenic progeny. Therefore, produce the restriction that transgenic animals are subjected to himself inefficient and unsettled transcript adjusting by pronucleus injection.

Homologous recombination technique in the ES cell has solved the potential difficult problem of most of pronucleus injections. Regrettably, up to now, there are two obstacles in its extensive use: restructuring is inefficient and except mouse any species are not all had available needed ES cell. One of problem of homologous recombination is that foreign DNA is inserted into the poor efficiency in the host chromosome. People's beta globin genes in the friend's cell is with 1 * 10^-7Frequency is directed to (Smithies etc., 1985). Provide about 6.3 * 10 with the construction guiding apoE locus that contains neomycin^-7Frequency (Piedrahita etc., 1992). Because low-frequency result, it is impracticable attempting to carry out homologous recombination by the pronucleus injection. Brinster etc. (1989) have reported that the occurrence frequency that carries out homologous recombination after direct pronucleus injection DNA enters mice embryonic is very low; By injecting more than 10,000 the pronucleus injection behind the embryo, mhc class ii E α gene single mutation (Brinster etc., 1989) is arranged.

Therefore, be the improvement gene by the homologous recombination success, must separate a carrier cell system. As mentioned above, set up the necessary carrier cell of transgenic animals and tie up to the existing description in mouse aspect. The described clone of using so far is ES cell and PGC or EG cell, because they are allowed manipulation in vitro and screen and produce then the transgenic animals that (by ES-or EG-Blastocyst injection) carries these changes. Continuous cell line (STO with the embryo fibroblast of the former generation embryo fibroblast (Wobus etc., 1984) of mouse or mouse; Ware and Axelrad, 1974) isolated the ES cell as feeder cells from the mouse embryo. In case isolate ES clone, they just can be maintained at undifferentiated state (Martin by cultivating at feeder layer, 1981), in culture medium, contain Buffalo rats'liver (BRL) cell conditioned medium (Smith and Hooper, 1987), or in culture medium, contain LIF ELISA (LIF; Smith etc., 1988; Williams etc., 1988). In addition, already shown by adding LIF and in cultivating system, might not only keep, but also when not having feeder layer, isolated ES cells system (Pease etc., 1990).

The clone that PGC derives is existing (Matsui etc., 1992 described in mouse; Labosky etc., 1994), its behavior is similar to ES clone and can works to described reproduction cell. When the separation of these cells is different from the separation of ES cell, by when LIF exists, cultivates at the MEC of deactivation and to make described culture remain on the mode same with the ES cell.

In mouse, progress crucial in present technique is embryonic derived clone or the evaluation of ES cell, described cell can carry out genetic manipulation external, is reintroduced to then among the embryo of growth with the formation to whole tissues of comprising the reproduction cell object of reference to work. Regrettably, no matter how big many researchers make effort, still can not isolate any ES clone with the reproduction cell feature (Stice and Strelchenko, 1995) from any species outside the mouse.

The existing report of the trial of exploitation ES and PGC technology in other mammalian species. Doetschman etc. (1988) show can separate ES clone from Hamster embryos with containing mouse embryo fibroblast of former generation. Separated hamster ES cell has the morphology that can be different from ES cells and the feature of vitro differentiation. Yet, repeated attempt howsoever, enter host embryo's blastocyst in injection after, can play a role to chimeric formation without any tested hamster ES clone. Although separated ICM is formed with effect to the chimera in same strain groups certainly, this technology still is used to detect described ES cell (Piedrahita etc., 1992).

Sukoyan etc. (1992) have reported the ES like cell system of mink, when it is injected into host's blastocyst, do not produce chimaera progeny. Yet as if because the result of fibroblast type is had in the teratoma test of using these identical cells to carry out, the potential of these cells is restricted. Regrettably, the different teratomatous researchs of cell type of later generation (Sukoyan etc., 1993) do not continue deeply to use the research of Blastocyst injection.

Existing ES like cell is in rabbit description (Graves and Moreadith etc., 1993; Giles etc., 1993). Newly separate or three days ICM cell of cultivation less than has effect to chimaera progeny, does not have such effect (Giles etc., 1993) and cultivate the ICM cell that surpasses three days. Recently, Du etc. (1995) has reported and has utilized rabbit ES like cell to carry out embryo after consideration convey moves to the growth in blastocyst stage. Yet general generally accepted is that it is not the good index that nucleus produces the offspring's who lives ability that blastocyst forms.

Present inventor and other people (Piedrahita etc., 1990; Evans etc., 1990; Stice and Strelchnko, 1996) use STO as feeder layer, reported the embryonic derived clone of pig of isolating ES sample morphology and limited vitro differentiation ability. Other people has also reported pig ES like cell system (Strojek etc., 1990; Notarianni etc., 1990; Talbot etc., 1993; Anderson etc., 1994; Wheeler, 1994; Gerfen and Wheeler, 1995). Be similar to the research to rabbit, Anderson etc. (1994) have shown with the new ICM cell that separates rather than with the ICM generation chimera of cultivating. Weeler (1994) has reported and has produced chimeric success. Yet the chimericization degree of not only reporting is low to not existing, and does not still have so far the report of the genotypic reproduction cell transmission of described ES aspect. In addition, when same experimental group can not repeat this as a result the time, described result causes people's suspection (Gerfen and Wheeler, 1995).

Wheeler (United States Patent (USP) 5,523,226) disclose and obtained to be used for to mix the pig embryo with the method for the embryonic stem cell that forms chimeric pig, be included in or when not having feeder layer, in the stem cell media of conditioning, cultivate embryonic stem cell, and confirm that described embryonic stem cell can form tumour in the SCID mouse. Yet the embryonic stem cell that Wheeler describes is different from the PGC cell, does not demonstrate and can transmit needed phenotype.

Hogan (United States Patent (USP) 5,453,357) discloses composition and the method that relates to the PGCs cell, comprises fibroblast growth factor, LIF ELISA, solubility steel factor and to keeping the very crucial film of PGCs in conjunction with steel factor. Yet, do not demonstrate the germ cell line transmission of described PGC phenotype.

The greatest difficulty of illustrating the document relevant with pig ES cell may be exactly the heterogeneity of separated clone, and described clone sometimes can be at external again tissue. Piedrshitaz etc. (1990) have reported the pig ES cell of isolating limited vitro differentiation ability. Although the structure that obtains is similar to capsule embryo individuality, optics and electron microscope histologic analysis show that this morphological change more can show again tissue than real differentiation.

Yet the great majority report uses the cyst data not have the basic evidence of any these statements of support as the indication of versatility differentiation always from that time. In addition, as from the cell outward appearances such as cell of the muscle of cultured cell system and neuron type by explain as described in clone versatility and to the homophylic indication of ES cell. The problem that this explanation brings is even also there is identical ability in the non-ES cell like cell system that is derived from body early embryo. And the cell that obtains from pig primitive ectoderm (can not produce the stage of ES cell mouse) breaks up external the experience widely. In addition, even detected clone is the ES cell really, still there is not the relation between the ability of reporting proof clone vitro differentiation ability and cloning chimera germ cell line.

Present inventor and other people (Shim and Anderson etc., 1995) have begun to utilize the suitable condition that mouse EG separates with the pig primordial germ cells that obtain from 25 days embryos and can isolate and keep typical ES morphology and express several clones that alkaline phosphatase (mark that does not break up the ES cell) reaches 14 generations and four months. Although, these cells produced chimeric ability and still belonged to the unknown this moment, present inventor's studies show that described morphology outward appearance and alkaline phosphatase activities feature (embodiment 2,3) arrive seen in the ES cells than any other previously described pig ES clone is more approaching. According to the experiment of making of mouse, the morphology outward appearance of described cell is that cell is to the good indicator of germ cell line ability to function.

Attempt by on the sheep skin fibroblast, when (Handyside etc., 1987) being arranged or do not have (Piedrahita etc., 1990) Buffalo rats'liver (PRL) conditioned medium, cultivate embryo's separation sheep ES cell and already failed. Notarianni etc. (1991) have reported the clone that the sheep ES of limited vitro differentiation ability derives, and Campbell etc. (1995) have reported the sheep nucleus shift experiment that carries out with the placenta cells that goes down to posterity in early days, and described placenta cells can instruct and develop into the offspring.

Cell and ES cell dissmilarity that Campbell etc. (1995) describe. Belong to epithelium on its morphological properties, and they do not express alkaline phosphatase. Although obvious generation offspring's ability is arranged, it be unclear that whether similarly method can be in other species (for example pig) effectively. This is because the sheep cell keeps the totipotency (even also like this at non-ES cell) of its nuclear of longer time than pig cell. The efficient that the cell that Campbell etc. (1995) separate produces the animal of becoming pregnant is extremely low, and this shows that the serviceability of these cells may be limited. In mouse, ES and EG clone can be maintained the long period and not affect the chimeric ability of its generation germ cell line.

Recently, Wilmut etc. (1997) has used Adult Mammals cell (mammal galandular epithelium) to produce the filial generation lamb that can survive. Yet the efficient of the method is very low.

Equally, use untreated STO as feeder cells, separating with limited successful report (Evans etc., 1990 only aspect the embryonic derived clone of the morphologic ox of ES sample; Stice and Strelchenko, 1996), move rear several epithelioid cell systems (Stice and Strelchenko, 1996) that embryonic development are formed with ability to function although reported consideration convey. Yet these clones can not produce offspring alive. The ES like cell of ox is existing (Saito etc., 1992 of announcing of other report of aspect; Stice etc., 1994). Sims has reported use with Firs (1993) as has not certainly had the embryonic derived clone of nucleus donor of separating in the suspension cultivation of feeder layer and can move acquisition offspring alive by consideration convey. Regrettably, no matter how initial researcher and other people make great efforts can not repeat these results always.

One group of researcher has reported the separator of the colony of 7 days ox PGCs of plating in two pieces of digests, they are alkaline phosphatase staining positives and have ES sample morphology (Cherny etc., 1994; Stokes etc., 1994). And injection FITC labeled cell enters host's blastocyst and shows the ability that is injected cell and described ICM cell aggregation. Yet, never further report, and do not learn that any cells involved participates in the data that chimera forms ability. Although these somewhat suspicious reports are arranged, so far, still have nothing to do at any non-rodent speciation report that does not break up transgenic cell line embryonic derived or PGC derived cell system.

Along with in ox and pig species, isolating the ES cell, inventor's research had determined to make described ES cell to maintain the condition of a bit of time of undifferentiated state already, but can not keep described clone (Moore and Piedrahita, 1997) when a kind of undifferentiated state is long enough to attempt genetic modification.

The next important step that produces transgenic animals is the multipotential cell that transforms with selected genes. In case these transformants are produced, the ability that they can the selected transgenosis of analyzed transmission give the filial generation animal. Yet although the report of many obvious versatility ES and PGC derived cell system is arranged, the conversion of non-rodent ES-or PGC-derived cell or clone does not still have report.

For detecting transformation cell lines to the ability to function of germ cell line, can use two kinds of technology; Be used for Blastocyst injection and nucleus transfer that chimera is produced. In pig, proved already that injection ICM entered blastocyst generation chimera pig (Onishi etc., 1994 of growth; Anderson etc., 1994). Therefore, the existing not yet sure report in ability aspect that works to producing chimeric pig about the ES like cell of cultivating. (Wheeler, 1994; Gerfen and Wheeler, 1995). Do not have the transmission of germ cell line, the hereditary change of any introducing ES cell can not be passed to the next generation and therefore, even described animal has real value also very little. Yet ICM can show to the fact that germ cell line works that if germ cell line can be maintained at undifferentiated state they should be able to show as ICM and by described germ cell line and carry described hereditary change.

Can be resolved by using consideration convey to move to walk around chimera to form to the problem that forms the separated embryo cell line that ES blastocyst germ cell line chimera (producing the prerequisite of the transgenic strain of animal) works. In the consideration convey shifting method, the nuclear of the cell of described correct modification is transferred in the ovum that enters stoning, and makes described embryonic development to the period of transferring to behind the suitable acceptor. Although the nuclear consideration convey of blastocyst stage embryo or ES cell moves the offspring that do not produce any work or mouse (Barnes etc., 1987 of pregnancy in mid-term; Robl etc., 1986), similarly method can be more successful ox and small-sized ruminant, because the underwriting of ox and sheep is being held totipotency, at least until blastocyst stage. This is confirmed (Smith and Wilmut, 1989 by the offspring's who obtains behind the enucleation oocyte that shifts the ICM nucleus and enter ox and sheep living ability; Keefer etc., 1994). And, proved already that the embryonic derived clone of ox can be used as nuclear donor, although (Stice etc., 1996 are lost in becoming pregnant of producing during the 3rd pregnancy period; Strelchenko, 1996).

As detailed above, still can not isolate allow with mouse in the same non-rodent ES cell of processing. After will allowing accurate processing genomic material, the availability of standing the PGC of check of the conversion that keeps undifferentiated state or EG clone produces the live animal of carrying those changes.

The invention provides the new method that produces the non-rodent of transgenosis. As detailed in this article, in certain embodiments, the present invention relates to the genitaloid method of culture of isolated, wherein said primordial germ cells are stood the check of conversion and are kept a kind of undifferentiated state.

In further preferred embodiment, separated primordial germ cells are converted before cultivation, and are converted cell and are used to produce the non-rodent of transgenosis at 1-3 after generation. In other preferred embodiment, cultivate the described primordial germ cells that are converted, the wherein said cell that is converted remains on a kind of undifferentiated state.

In further preferred embodiment, with providing the DNA section of homologous recombination to transform described primordial germ cells. In some other preferred embodiment, selected genetically modified flank is to promote under proper condition excision to be impregnated in genetically modified dna sequence dna.

1. embryonic cell

Separation has unlimited multiplication capacity under undifferentiated state from cytoplasmic embryonic stem cell within the PIE, can break up in vitro and in vivo, and can work to the normal structure of chimeric individuality and the formation of organ when being administered to the host embryo. Differentiation can stimulate by revising condition of culture, stimulates (Doetschman etc., 1985) and enter athymic mouse by injection ES cell in vivo. When allowing in vitro differentiation, the ES cell forms the structure that is called embryoid, and this structure extremely is similar to the idiosome part (Doetschman etc., 1985) of mouse in 5 day age.

The ability of cultivation and the described reproduction cell of genetic manipulation rear cloneization has made the ES cell become the tool of modifying mouse genome. The chimera that produces between genetic modification ES cell and the fetal tissues has been used to study the reproduction cell transmission (Smithies, 1991) that vivo gene is regulated (Stewart etc., 1985) and is introduced into gene. In addition, the ES cell has been used to study gene directed modification (Smithies, 1991 of being undertaken by homologous recombination; Piedrahita etc., 1992).

Although the majority research to the ES cell is carried out, have numerical digit researcher's report in the trial of other mammalian species exploitation ES technology in mouse. Doetschman etc. (1988) demonstration uses the feeder cells that contain mouse embryo fibroblast of former generation to separate the ES cell from Hamster embryos. Numerical digit researcher uses STO to do feeder layer, has reported the embryonic derived clone of pig (Evans etc., 1990 of isolating the limited differentiation capability in ES sample morphology and external and the body; Notarianni etc., 1990; Piedrahita etc., 1990; Strojek etc., 1990; Gerfen and Wheeler, 1995). Therefore, in pig, proved that not only injection ICM enters blastocoele generation chimera pig (Anderson etc., 1994 of the blastocyst of growth; Onishi etc., 1994), and existing report, the ICM-of the cultivation ES like cell of deriving has the ability (Wheeler, 1994) that produces the chimera pig. Yet the degree of chimericization of not only reporting is low, and does not still have so far report (Wheeler, 1994 of the genotypic germ cell line transmission of ES aspect; Gerfen and Weeler, 1995). Do not have the transmission of germ cell line, any hereditary change of having introduced described ES cell all can not be passed to the next generation, thereby, even this animal is valuable also little.

Recently, existing report, the performance of the mouse cell line that primordial germ cells are derived is similar to the ES cell and can work to described germ cell line (Labosky etc. 1994). (Labosky etc. 1994 for these cells (being called EG cell or PGC-derived cell); Strel chenko, 1996) aspect the sign of described undifferentiated state and after injection enters host's blastocyst, make the ability of described germ cell line cloning be similar to ES cell (Labosky etc., 1994; Stewart etc., 1994). Therefore, be that in case be established, they just have similar character even initial structure source or cell phenotype are different from described ICM-derived cell. Shim etc. (1997) had reported that PGC derived cell system to forming the chimeric ability to function of pig, had proved the versatility feature of these clones. The inventor has enlarged above-mentioned observation by the PGCs of proof genetic transformation to the ability to function that chimera forms, shows that cell of the present invention has the versatility feature and shows that described genetic transformation and system of selection do not affect described cell and participate in the ability that chimera forms.

The result who obtains with PGC (EG) derived cell system shows that they have more chance that transgenosis is modified with usefulness than embryonic derived ES cell. Its reason comprises: isolate 10,000 to 20,000 genitaloid abilities (Shim and Anderson, 1995 from single embryo; Piedrahita and Bazer, the disclosure), for the separation of ES cell, each embryo on average only is 12-15 cell by contrast; Obtain to have the ability that multipotential cell is the cell colony of morphology and typical cells mark of not breaking up from described PGCs high-frequency; Keep and transmit the ability of described PGC cell colony to the time that is enough to allow genetic modification; PGC clone is to the ability to function of described chimera germ cell line; With the potential use of EG cell as the nucleus donor of embryo cloning research.

Show that with the PRELIMINARY RESULTS of pig inner cell matter injection versatility EG cell enters the embryonic development blastocoele and has a good chance and transmit hereditary change by germ cell line. Use the consideration convey of the EG clone of ruminant species to move to be that to use these embryos to carry out the technological merit that consideration convey moves research be the basis. So far, still can not move research from the consideration convey of the pig that surpasses 8 cell stages and obtain any filial generation (Niemann and Reichelt etc., 1993). Except a few exceptions (Machaty etc., 1996), also can not develop and can be used for producing ovum and be suitable as a kind of external maturation of ovum system (IVM) that consideration convey moves acceptor.

On the contrary, the research of carrying out in ox has shown that the nuclear of 7 days embryos' inner cell matter still can develop into complete organism (Keefer etc., 1994) after consideration convey moves. Therefore, IVM, the technology of IVF is well developed in ox. Equally, Campbell etc. (1996) has recently reported from the sheep embryo derived cell system of going down to posterity after cultivating for 13 times and has produced efficient offspring. Because it is similarly (Smith and Wilmut, 1989 that consideration convey moved on the time that research shown already that sheep and Niu Zaiqi totipotency lost; Keefer etc., 1994), as if the performance of the embryonic derived cell of ox of cultivating can be similar to the performance of sheep embryo derived cell.

A. the embryo separates

Embryo collection is from the screened non-rodent species jenny of becoming pregnant. Described animal is anaesthetized (and being removed the uterus) or described embryo can collect after butchering. The embryo is collected very early gravidic usually. For example, the pig embryo was collected in gestation in 25 days, and the ox embryo was collected at pregnant 35-40 days, and sheep and he-goat embryo collected oestrus 6 or 7 days.

B. separate and cultivate primordial germ cells and be

In case the embryo is collected, just separate primordial germ cells (PGCs). Primordial germ cells are multipotential cells, and it has the ability that is divided into whole three primordial germ cells layers. In mammal, described PGCs is from allantois substrate migration by rear enteric epithelium and dorsal mesentery and cloning germarium primordium (Eddy etc., 1981). The PGC derived cell has cytoplasm low on the feature/nucleus ratio, and major part is nucleus usually. The separation method of described PGCs is the sex-ridge that shifts out the embryo, and PGCs is broken away from from germarium primordium, and collects described PGCs. Some reports that PGCs can be frozen storage, and thawing and cultivating had 60% survival rate (Leichthammer and Brem, 1990) in rear 24 hours. The pig PGCs of stored frozen also can carry out consideration convey and move (Liu etc., 1995).

With regard to the use among the present invention, primordial germ cells use when collecting, or use in 24 hours at the PGCs of the stored frozen of thawing. Described PGCs can be directly used in conversion, maybe can be placed under proper condition to cultivate described cell. The present invention discloses improved condition of culture, and it increases PGC colony number and reaches 5 to 10 times under existing system. Even after repeatedly going down to posterity, the expression of the morphology of described colony and their alkaline phosphatase extremely is similar to the inner cell matter of new plating. Never observe this differentiation with ICM derived cell system and suppress phenomenon (Piedrahita etc., 1990).

1. feeder cells

Isolated primordial germ cells are cultured on the feeder cells. The spendable feeder cells type of the present invention is such as mouse SI/SI⁴Embryo cell line or the embryo fibroblast of separated animal species, such as pig or ox. What more preferably use in the present invention is STO cell (MEC; Ware and Axelrad, 1972). Aspect some, only express the SI of the stem cell factor of film combining form of the present invention⁴-m220 cell can be used. Described feeder cells provide the primordial germ cells of growth factor to growth, but the Endogenous Growth Factors quantity that provides is all not identical in each preparation. Therefore, can add the growth factor of external source adding to replenish endogenous supply. In addition, some concrete aspect of the present invention, the present inventor expects that the feeder cells of through engineering approaches can express selecteed growth factor, for example film is in conjunction with stem cell factor and basic fibroblast growth factor.

Described feeder cells are inactivated before use, preferably use the X ray of reagent such as cobalt or caesium, or use mitomycin C. The feeder cells of deactivation are cultivated, preferred 24 hours, but longer and shorter incubation time also is fine.

The feeder cells density that is inactivated to of the present invention successfully be crucial. The preferred density of using is about 1.5 * 10 in the present invention⁵With 10⁶Between the cells/square cm, preferred density is about 2-4 * 10⁵Between the cells/square cm. This external some aspect of the present invention was preferably used per 3 to 5 days, and per 35 millimeters holes add 1-1.5 * 10⁶New feeder cells.

2. supporting base forms

The invention provides the composition for the culture medium of primordial germ cells growth. Described PGCs, when thawing from stored frozen or transforms on the rear feeder cells that are inactivated of directly cultivating in culture medium at after separating. Preferably making culture medium used in this invention is the improved Eagle culture medium of LG Dulbecco. Ham F10 culture medium further preferably. The bond of the improved Eagle culture medium of LG Dulbecco (about 50%v/v) and Ham F10 culture medium (about 50%v/v) more preferably. Described medium optimization replenishes Glu. Preferred culture medium replenishes with the β mercaptoethanol in addition, and also has other preferred culture media supplemented 100nM nonessential amino acid (L-Ala, L-Asn, L-Asp, L glutamine, glycine, L-PROLINE and L-Ser; GIBCO). More preferably making used in this invention is complete supplementing culture medium, comprises in addition growth factor below one or more.

a.bFGF

An essential culture medium constituent of using among the present invention is basic fibroblast growth factor (pFGF). BFGF is a member of FGF family, finds at present to comprise nine relevant short split proteins that the amino acid conservative is 35-55%. Different from other member of great majority of its described family, bFGF does not have signal peptide and obviously secretes with the mechanism of non-traditional protein secretory pathway. BFGF can be separated to from many sources, comprises nerve fiber, hypophysis, adrenal cortex, corpus luteum and placenta. BFGF contains four cysteine residues and reduced form bFGF has kept completely BA. Several reports show that various forms of bFGF are that prolongation owing to the N end generates. These continuations obviously affect bFGF in lumen the position but do not affect BA. Recent studies show that be essential with the combination of heparin or cell surface heparin sulfate proteoglycans to the combination of FGF and high affinity FGF acceptor.

BFGF stimulates cell and many neuroderms in whole mesoderms source, the propagation of the cell in ectoderm and entoderm source. Described cell comprises fibroblast, endothelial cell, star-like cell, oligodendrocyte, neuroblast, horn cell, Gegenbaur's cell, smooth muscle cell and melanocyte. For external endothelial cell, bFGF is chemotactic and short fissional. BFGF induces Neural Differentiation, survival and regeneration. BFGF also can demonstrate the key effect in regulation of embryonic development and differentiation. BFGF observation in vitro to these function prompts bFGF can play in vivo such as Angiogenesis wound healing and tissue repair, the regulating action of embryonic development and differentiation and the normal processes such as neuronal function and neurodegeneration. In addition, bFGF can participate in producing cell hyperproliferation and the excessive various pathological conditions brought of sex change.

N not 146 flat amino acid whose isoform people bFGF of end-grain cutting is cloned (Abraham etc., 1986). The recombination human basic fibroblast growth factor of expressing in E.coli is bought (goods Article Number 233-FB) from R﹠D Sysrems. The present inventor also expects to be used for the present invention (SectionVIII) from selected animal species clone basic fibroblast growth factor.

B. uterus ferritin

The uterus ferritin is purple, progesterone induced contain two iron molecule glycoprotein, by endometrial epithelium secretion (Bazer and Roberts, 1983 of pig; Roberts and Bazer 1984). The uterus ferritin is with 35, the form of 000M polypeptide exists, and is purple, and becomes a kind of heterodimer (M=80 with one of the amino acid sequence that the serpin homology is arranged three kinds " uterus ferritin GAP-associated protein GAPs ", 000) (Murray etc., 1989). Described heterodimer has a kind of rose-colored, but biochemistry and the biological significance of the uterus ferritin of this rose form and uterus ferritin related protein still belong to the unknown. The uterus ferritin carries the high mannose carbohydrate, contains the identification marking Man-6-P (Baumbach etc., 1984) of lysosomal enzyme and activity of acid phosphatase (Schlosnagle etc. 1974) is arranged. Period of gestation, by being called the specialization placenta structure of nidulus, the uterus ferritin is secreted the blood circulation that is delivered to embryo's placenta (Renegar etc., 1982) from the uterus. Mannose residue on the ferritin of uterus and uterus ferritin are directed to the reticuloendothelial cell (the main position that haemocyte generates in the embryo pig) relevant (Sauders etc., 1985) of described embryonic liver.

Using radiolabeled iron to pig causes endometrial secretion to carry the uterus ferritin of radioactive iron and makes radiolabeled iron mix tire red blood cell and liver, the cell of spleen and marrow (Ducsay etc., 1982,1984). The uterus ferritin discharges its iron to the embryo's transferrin in the allantoic fluid, and the half-life is 12 to 24 hours (Buhi etc., 1992). And during the ferritin maximal secretory capacity of endodermis uterus, the pig iron administration glucan of giving conceived 50,60 and 70 days causes iron reserves rising 20% (Ducsay etc., 1982,1984) in the newborn piglet. These results suggest uterus ferritin effects in striding the placenta iron transfer. Yet, after conceived 75 days, the mRNA translation rapid decrease (Simmen etc. of uterus ferritin, 1988), the uterus ferritin secretion decline (Basha etc. that the endometrium explant is cultivated, 1979), and the amount of the uterus ferritin in the allantoic fluid sharply descend (Basha etc., 1975). This show another kind stride placenta iron transfer mechanism at the 75th day and embryo/placenta to begin to work during in the increase in demand of iron (Ducsay etc., 1982,1984).

The uterus ferritin in pig uterus is the acid phosphatase of tartrate resistance, with (Ketcham etc. among the people embryo, 1985), among osteoclastoma patient's the cartilage cell, the trichoblast leukaemia, the total numerous characteristics of 5 type acid phosphatases in Gaucher disease and Hodgkin patient's the spleen. In addition, the feature class of uterus ferritin is similar to ox, rat, mouse, and pig spleen, and cow's milk, cattle uterus secretion, the feature of the purple acid phosphatase in nightstool palace secretion and the Rat Skeletal (Ketcham etc., 1985).

Uterus ferritin and uterus ferritin rose have demonstrated the propagation (Bazer and Gross, United States Patent (USP) 5,258,367, this paper are cited as reference) that helps the hematopoietic stimulation cell. Uterus ferritin and uterus ferritin rose affect the differentiation of non-adhesion candidate stem cell of former generation in non-species specificity mode.

Uterus ferritin and rose can obtain by various distinct methods. These examples can from pig the uterus flush obtain (Baumbach etc., 1984; Murray etc., 1989) or the allantoic fluid of false pregnancy pig obtain (Baumbach etc., 1986). People uterus ferritin claims again people's placenta V-type acid phosphatase, can be such as the description purifying of (1986) such as C.M.Ketcham. The uterus ferritin also can produce by recombinant technique (Simmen etc., 1988; Ketchman etc., 1989).

C. the relevant SCF of film

It is believed that the feeder cells that are inactivated provide the film related stem cells factor (SCF) for primordial germ cells. The relevant SCF of film lacks the exon 6 of encoding proteins enzyme cleavage site. Provide the feeder cells of the relevant SCF of film to can be used for some aspect of the present invention. The relevant SCF of the preferred also promising overexpression film that uses or only for expressing the be correlated with feeder cells of SCF through engineering approaches of film in some aspects.

D. solubility SCF

Solubility stem cell factor (SCF) is another kind of growth factor, and it can be used in the specific embodiments of the present invention. SCF be the known PGC of being conducive to Motility with (or) cell factor of propagation. SCF decrease PGC cultivates Apoptosis (cell death of the sequencing) incidence (Pesce etc., 1993) in the first few hour. The C-kit part that is accredited as recently the part of kit tyrosine kinase receptor is positioned in mouse S1 locus. This multiple-effect cell factor can be called stem cell factor (SCF) in addition, mast cell growth factor (MGF) and steel factor (SLF), and it forms at gamete, and the commitment that melanocyte forms and haemocyte forms plays requisite effect. In vitro and in vivo, SCF can stimulate ripe proliferation of mast cells, and the propagation of immature mast cell and maturation. To the former generation people of purifying and the hematopoietic cell precursor of mouse, SCF with various growth factors such as IL-1, IL-3, IL-6, the synergistic mode of IL-7 and erythropoietin(EPO) works, inducing marrow, red blood cell and lymphocyte pedigree cell colony form. The discovery prompting SCF that SCF also expresses in nervous system may work in nervous system development.

The people, the cDNA coding transmembrane protein of Mouse and rat SCF comprises a signal peptide (a kind of 189 amino acid whose cell foreign lands), a hydrophobic transmembrane territory and a cell internal area. Natural SCF can film combining form or the soluble form that forms with front 164 or 165 amino acid by the cell foreign lands exist. Described soluble form is considered to the protein cleavage product of transmembrane protein. Described solubility all has growth factor activity with the SCF that strides form membrane. Natural soluble SCF is height N-and the glycosylated protein of O-, and its binary form with non-covalent combination exists in solution. Whole four cysteine residues of SCF monomer are relevant with disulfide bond in the cell. It is BA that the recombinant soluble SCF that produces in E.coli detects at external biological, and pointing out the glycosylation of described soluble form is nonessential for Bioactivity. Mouse or soluble rat SCF and people's solubility SCF has high homology (about 80%). And rat and mouse SCF all are effective to people's cell, and described human protein is much smaller to the effect of mouse or rat cell.

Be encoded into acquaintance SCF protein DNA sequence and be cloned (Martin etc., 1990). Recombinant human scf from E.coli can buy from R﹠D Systems (goods Article Number 255-SC).

e.LIF

Spendable another somatomedin is leukaemia inhibitory factor (LIF) in certain embodiments of the present invention.LIF is another kind of cytokine, and it also shows by reducing the survival (Pesce etc., 1993) that apoptosis promotes PGC.Leukaemia inhibitory factor (LIF) is accredited as inhibition propagation at first and induces to the mouse bone marrow cells leukemia cell is a kind of factor of M1 scavenger cell differentiation.Along with its purifying and molecular cloning, LIF is identified as all has the polyphenic multiple-effect factor to hematopoiesis and non-hematopoietic cell.LIF has OSM, IL-6, IL-11 and CNTF eclipsed biological function.All these cytokines are utilized the composition of gp130 as their signals conduction receptor complex.

The people LIF cDNA polypeptide that has 202 amino-acid residues of 22 amino-acid residue signal peptides of encoding, its cracking produces the one-tenth acquaintance LIF of one 180 amino-acid residue.The LIF of natural human and mouse is the monomeric protein of high glycosylation.People and mouse LIF protein sequence all have glycosylation site that multipotency N-is connected with O-with three six conservative cysteine residues that intramolecular disulfide bond is relevant.Nonglycosylated, E.coli expresses, and recombinant human LIF is being different from natural LIF aspect the extracorporeal biology activity.The ripe LIF of people and mouse presents the sequence identity of 78% amino acid levels.And people LIF has same function to people and mouse cell, and mouse LIF hangs down 1000 times to people's cell approximately to effect.

The recombinant human LIF that expresses in E.coli can be from R﹠amp as a kind of fusion rotein of and glutathione s-transferase (GST) (cut and carry out the HPLC purifying from GST); D Systems (goods Article Number 250-L) buys.Present inventor's expectation is from corresponding animal species (VIII joint) clone LIF.The present inventor has optimized and has been used for known pig LIF (the SEQ ID NO:7 that expresses at yeast when the relevant LIF of the animal species that uses self-sizing draws to an end with the work that produces PGCs; The V joint).

F. apoptosis supressor

Numerous protein has demonstrated the inhibition apoptosis, i.e. the necrocytosis of sequencing.Because shown already that suppressing apoptotic somatomedin promoted primordial germ cells survivals (Pesce etc., 1993), this proteinoid is particularly preferably in using among the present invention.Alpha2 Macroglobulin is the particularly preferred apoptosis supressor example that uses aspect some in the present invention.This class representative also has oncogene protein such as bcl-2 and comprises Bcl-x1, Mcl-1, Bak, A1, the family member of A20, and interleukin-1 ' beta '-conversion enzyme and kinsfolk.What preferably use is that bcl-2 (with bcl-1, distinguish by cyclin D1; GenBank Accession No.M14745, X06287).The overexpression of this oncogene is at first found in t cell lymphoma.It works by combination and deactivation bax (a kind of protein in the apoptotic pathways) as oncogene.

Many other factors are supposed to prevent according to its blocking-up, or reduce apoptotic ability and use in culture media composition of the present invention.As when interleukin 3 (IL-3) when the IL-3 dependent cells shifts out, calcium ion carrier A 23187 presents the blocking-up apoptosis in some system.N-acetyl-L-cysteine has demonstrated the apoptosis (Ferrari etc., 1995) that prevents neurocyte, and TNF-α induces the apoptosis (Cossarizza etc., 1995) of U937 cell.Nakajima etc. (1994) show that dactinomycin (although are a kind of potential inductors in many clones) has demonstrated the programmed cell death that suppresses etoposide (a kind of inhibitor of topoisomerase II) inductive PC12 cell.These researchs also show Cyclohexamide, and nerve growth factor and Urogastron are also brought back to life the PC12 cell from the death of etoposide inductive.RhIGF-1 (IGF-1) and described IGF-1 acceptor also demonstrate and suppress etoposide inductive apoptosis (Sell etc., 1995) in the BALB/c3T3 cell.

3 aminobenzamides have shown as the apoptotic inhibitor of UV inductive (Malorni etc., 1995).The aphid rhzomorph of dwelling promotes in the leukemia cell system to be suppressed by pectinose nucleosides inductive apoptosis vincristine(VCR) inductive apoptosis (Borner etc., 1995) among the negative PC-3 PC-3 of p53.Xitix (vitamins C), catalase, follicular stimulating hormone, N-acetyl-L-cysteine, vasoactive intestinal peptide, cyclo GMP, hCG, interleukin-1 ' beta ' (IL-1 β) and superoxide-dismutase all show as and suppress or check apoptosis (Flaws etc., 1995 in the rat ovary ovarian follicle of cultivation; Tilly and Tilly 1995; Chun etc., 1995).The golden red tricarboxylic acid has shown as inhibition by the apoptosis (Benchokroun etc., 1995) in the various cellular types of various factor inductive

BAPTA/AM[1, two (the adjacent ammonia phenoxy group) ethane-N of 2-, N, N ', N '-tetraacethyl four (ethoxyl methyl) ester] suppress thapsigargin inductive apoptosis (Jiang etc., 1994) in the rat chest cell.Caffeine has shown and has prevented camptothecine and topotecan inductive apoptosis and cell cycle effect (Traganos etc., 1993) in the HL-60 cell.Calpain inhibitor I suppresses the apoptosis (Squier etc. in thymocyte and the metamyelocyte, 1994), and leupeptin (calpain inhibitor II) and E64 class serpin have also shown the programmed cell death (Sarin etc., 1994) that suppresses activation-inducing.Cyclosporin A has shown the apoptosis that prevents anti-Ig-M and ionomycin inductive BLB clone.

General serpin 3,4-dichloro Isocoumarin 〉97 and specificity thiol reagent N-ethyl maleimide show dna break (Cain etc., 1994) between the apoptotic nucleosome that no endonuclease participates in the blocking-up thymocyte.Cysteine protein matter inhibitor E64 and leupeptin, calpain selective depressant acetyl-leucyl-leucyl-positive first two sulphur aldehyde, all show mouse T cell knurl and the programmed cell death in the activated periphery T cell (Sarin etc., 1993) that the selective exclusion TXi Baoshouti causes with serpin diisopropylfluorophosphate and phenylmethylsulfonyl fluoride.Tetraodotoxin, nimodipine, isoptin, flunarizine and R56865 all protect the ox pheochromocyte to avoid veratridine inductive necrocytosis (Maroto etc., 1994).

Hair monkey element and insulin-like growth factor-1 (IGF-1) all show the apoptosis that suppresses cerebellar myeloid, although carry out (Galli etc., 1995) by different mechanisms.Protein tyrosine kinase inhibitor genistein and Antibiotic TAN 420F all show and prevent to resist-CD3 monoclonal antibody inductive thymocyte apoptosis (Migita etc., 1994).Interleukin 6 (IL-6) suppresses the composing type of mouse B cell hybridoma 7TD1, protein synthesis dependent/non-dependent apoptosis (Liu etc., 1994).Apoptosis (the Gjertsen etc. of the glucocorticoid inducible of protein phosphatase inhibitor calyculin A and okadaic acid suppressor T cell hybridoma, 1994), and the known apoptosis that prevents gamma-rays inductive Burkitt lymphoma cell line BM13674 of calyculin A.

Protein kinase C activation agent phorbol-12-myristic acid-13-acetate suppresses the apoptosis (Tepper etc., 1995) of Fas antigen induction.1-tetramethyleneimine dithionic acid prevents the apoptosis (Bessho etc., 1994) of human promyelocytic leukemia HL-60 cell and thymocyte.Calcium channel blocking-up thing NIFEDIPINE and nisoldipine and endonuclease enzyme inhibitors golden red tricarboxylic acid have shown the apoptosis (Escargueil-Blanc etc., 1997) of the human endothelial cell of blocking-up cultivation.Spermine has shown and has suppressed the morphology apoptosis, and antioxidant sulphur oxygen cyclase protein suppresses the apoptosis (Sata etc., 1995) of JurkatT cell and people PBL blastocyst.In addition, proteinase inhibitor N ^α-tolylsulfonyl-L-phenylalanine chloromethane ketone, N ^α-tolylsulfonyl-L-Methionin chloromethane ketone and than the N of poor efficiency ^α-tolylsulfonyl-L-arginine methyl ester suppresses the apoptosis (Bruno etc., 1992) of thymocyte.

3. culture condition

The primordial germ cells culture is the optimum condition such as the pH of high growth, carbon dioxide percentage, and it is that those skilled in the art are well-known that oxygen is pressed.Preferred primordial germ cells culture condition is at 38 ℃, in the damp atmosphere of about 5% carbonic acid gas.

C. primary cultured cell analysis

With regard to using among the present invention, the primordial germ cells of described cultivation must remain on undifferentiated state.There are many methods to be used to measure whether cell is in undifferentiated state.At present, these methods depend on the expression of some marker of (negative screening) that (positive-selecting) that morphocytology or undifferentiated state are exclusive or differentiation state are exclusive.Undifferentiated morphocytology generally is the cell of the high nuclear-cytoplasmic ratio rate that closely fills up.And, in undifferentiated cell, exist significant kernel usually.

Preferred screening method also uses in the present invention.The method of the existing undifferentiated cell of a kind of preferred screening is undertaken by the screening alkaline phosphatase activities.Research shown expression of ALP and have between differential period good relationship (Talbot etc., 1993a, 1993b).The preferred method that uses among the present invention also has screening stage specificity embryonal antigen 1 (SSEA-1), and this antigen is PGCs (Donovan etc., 1986) and does not break up ES and the positive-selecting feature of ES cell (Solter and Knowles, 1987).The preferred negative screening of using among the present invention is a screening cytokeratin 18, and it is indication (Piedrahita etc., 1990 that cell is in differentiation state; Van Stekelenburg-Haers etc., 1995).

The conversion of II primordial germ cells derived cell system

In some preferred embodiment of the present invention, the described genetically modified nucleic acid of encoding can be stabilized and be incorporated in the described cellular genome.And in selecting a step embodiment, what described nucleic acid can a kind of isolating additive type DNA section be stabilized remains in the described cell.The nucleic acid of this nucleic acid segment or " additive type " coding is enough to allow and does not rely on or be synchronized with the described host cell cycle and Maintenance and Replication.The type that employed transgenosis construct is depended in the mode of cell and position that described nucleic acid keeps is advanced in described transgenosis construct transmission in described cell.

A. method for transformation

Be gene constructs of effective expression, described expression constructs must be passed in the primordial germ cells.As described below, the preferable mechanism of transmission is by electroporation, and calcium phosphate transforms or the particle bombardment method.Yet, the present invention also detailed consideration several transmit transgenosiss and enter genitaloid other method.In one embodiment of the invention, described transgenosis construct can only be made up of naked recombinant DNA or plasmid.The transmission of described construction by mentioned from physics or chemically any method of the described cytolemma of saturatingization carry out.

1. electroporation

In certain embodiments of the invention, transgenosis construct is introduced into described primordial germ cells by electroporation.Electroporation comprises makes cell suspension contact high voltage electric with DNA.

With the electroporation transfecting eukaryotic cells has been quite successful.The κ immunoglobulin gene transfection (Potter etc., 1984) of having chosen of mouse pre-B lymphocyte, and rat hepatocytes is with chloramphenicol acetyl transferasegene transfection in this way (Tur-Kaspa etc., 1986).

The genitaloid electroporation conditions of expectation different sources can be optimised.Can specifically use as voltage, electric capacity, the parameter of time and electroporation medium composition and so on is optimized.Other conventional implementation method of adjusting should be that those skilled in the art know.

Former generation the pig primordial germ cells and subculture (cultivation) pig primordial germ cells transformed (embodiment 3) with the electroporation success.

2. particle bombardment method

The present invention transmits one of preferred embodiment that the naked DNA construction enters cell and comprises the particle bombardment method.This method depends on that accelerated packet is bound to by DNA little to be made little bullet permeates cell membranes at a high speed and enters cell and the ability of cell killing (Klein etc., 1987) not.Employed little bullet is made up of as tungsten any biological inert material, platinum or gold bead.

Expect that DNA is deposited on the metallics making the alpha bombardment method transmit DNA to recipient cell not necessarily under some situation.The expectation particle can contain DNA rather than wrap quilt with DNA.Therefore, infer that the particle of DNA bag quilt can improve the level of DNA by the transmission of particle bombardment method, but be not that (portion or on its surface) within it is necessary.

The several devices of quickening small-particle develops.Wherein a kind of device produces electric current by high voltage electric, and the latter provides power (Yang etc., 1990).Other method comprises that the particle that uses BiolisticParticle Delivery System, this system and device can be used to advance the bag quilt DNA by a screen, as stainless steel or Nytex screen, arrives the filter surface that is coated with cell in suspension.Described screen disperses described particle so that it is not delivered to recipient cell with big group.Every the screen between the described projectile device and the cell that will be bombarded be considered to reduce projectile group size and can be by reduce the transformation that damage that too big projectile causes plays upper frequency on recipient cell.

With regard to blast technique, the cell in the suspension preferably concentrates on the filter, or on solid medium.The cell that will be bombarded is placed on little suitable distance under the stop board of launching.If need, between the booster machinery and the cell that will be bombarded, also can place one or more screens.

In bombardment transforms, can make preceding culture condition of bombardment and bombardment parameter optimization to produce stable conversion of maximum number.Physics and biological parameter all are important in this technology.Physical factor is to comprise the parameter of handling the sedimentary factor of the little bullet of DNA/ and influencing range and the either large or small projectile of speed.Biological factor is included in the preceding Overall Steps relevant with just bombarding the aftertreatment cell of bombardment, for assisting to alleviate the adjustment of the relevant wound of bombardment to target cell, also comprises the character of described transfering DNA, as linearizing DNA or complete super spirial plasmid.It is believed that the bombardment pre-treatment is crucial to successfully transforming primordial germ cells.

Therefore, the expectation experimenter can wish to adjust various bombardment parameters in the small scale experiments with the described condition of abundant optimization.The experimenter can wish to adjust physical parameter such as clearance distance especially, range, and tissue distance and helium are pressed.The experimenter also can make described wound reduction factor reach best by the physiological status of improving the described recipient cell of influence thereby the condition that can influence conversion and integration efficiency.For example, the osmotic pressure state of described recipient cell is organized degree of hydration and go down to posterity cultivation stage or cell cycle to be adjustable to optimize and is transformed.Other conventional implementation method of adjusting should be that those skilled in the art know.

3. virus transforms

A. Adenovirus Transfection

A method transmitting described transgenosis construct comprises uses a kind of adenovirus expression carrier.Though the ability that known adenovirus carrier is incorporated in the genomic dna is low, this characteristics are compensated by the high gene transfering efficiency that these carriers provided." adenovirus expression carrier " means those constructions that comprise the gland-containing virus sequence, and described sequence is enough to (a) and supports the packing of described construction and (b) finally express the transgenosis construct of wherein being cloned.

Described carrier comprises the genetically engineered form of adenovirus.The knowledge of heredity weave construction or adenovirus (a kind of 36kb, linear, double-stranded DNA virus) aspect makes and can replace big section adenovirus DNA (Gruhuas and Horwitz, 1992) up to the exogenous array of 7kb.Compare with retrovirus, the adenovirus infection of host cell does not cause chromosomal integration, because adenovirus DNA can duplicate in the episome mode and not have latent gene toxicity.And, be stable on the adenovirus structure, and constantly do not detecting genome rearrangement after the amplification.

Adenovirus is specially suitable for being used as gene transfer vector, because its medium sized genome, easy handling, high titre, broad target cell scope and high infectivity.Described virus genomic two ends contain 100-200 base pair inverted repeats (ITRs), and they are cis factors, by virus replication and the packing necessary.Genomic morning (E) with evening (L) district contain different transcription units, they begin to duplicate by viral DNA and are separated.Described E1 district (E1A and E1B) coding and viral genome transcriptional regulatory proteins associated matter and small amounts of cells gene.Described E2 district (E2A and E2B) causes the synthetic of viral dna replication desired protein.These protein and dna replication dna, late gene expression is closed relevant (Renan, 1990) with host cell.The product of described late gene comprises most viral capsid proteins, is only expressed after the obvious processing of single primary transcript that major late promoter (MLP) produces.Described MLP, (being positioned at 16.8m.u.) is highly effective late between period of infection, and has 5 '-three fens guide (TPL) sequences from whole mRNA of this promotor generation, makes it become the mRNA sequence of preferred translation.

In present system, recombinant adenovirus produces from the homologous recombination between shuttle vectors and provirus carrier.Because possible reorganization between two kinds of virus vector, wild-type adenovirus can produce from this process.Therefore, mono-clonal and its genome structure of detection from single plaque isolated viral is crucial.

The generation of current adenovirus carrier (it is a replication defect type) and propagation depend on distinctive synergid system (being called 293), and it is the also constitutive expression E1 protein (Graham etc., 1977) that transforms by the Ad5 dna fragmentation from the human embryonic kidney cell.Because (Jone and Shenk, 1987) can be removed from described adenoviral gene group by the E3 district, current adenovirus carrier (under 293 cells are assisted) at E1, carries foreign DNA (Granham and Prevec, 1991) in D3 or both zones.At occurring in nature, adenovirus can be packed about 105% described wild type gene group (Ghosh-Choudhury etc., 1987), and about 2kb is provided above extra DNA capacity.Be combined in commutable about 5.5kb DNA in described E1 and the E2 district, described current adenovirus carrier maximum capacity is about 7.5kb, or described carrier total length about 15%.Adenoviral gene group more than 80% remains in the carrier frame.

Synergid system can be derived from human cell such as HEKC, myocyte, hematopoietic cell or other people's embryo mesenchymal cell or epithelial cell.Perhaps, described synergid can be derived from the cell of the mammalian species of other energy receiver adenovirus.For example this type of cell comprises Vero cell or other monkey embryo mesenchyme or epithelial cell.As mentioned above, preferred synergid is to be 293.

Recently, Racher etc. (1995) discloses the modification method of cultivating 293 cells and replicative adenovirus.In a kind of mode, the n cell aggregation cultivate in the 1 liter of silanization turn flask that contains 100-200 milliliter substratum by inoculating each cell (Techne, CambridgeUK).After the 40rpm stirring, estimate described cell survival rate with Trypan Blue.In another form, by following use Fibra-Cel microcarrier (Bibby Sterlin, Stone, UK) (5 grams per liter).A kind of be resuspended to 5 milliliters of cell inoculation things in the substratum be added into 250 milliliters in the Erlenmeyer flask carrier (50 milliliters) and left standstill (once in a while stir) 1 to 4 hour.Described then substratum is replaced and the beginning jolting with 50 milliliters of new substratum.With regard to virus produces, make cell grow into about 80% and be paved with, after this change described substratum (to 25% final volume) and add adenovirus with 0.05 MOI.The culture standing over night increases described volume to 100% then and began jolting other 72 hours.

Except described adenovirus carrier is a replication defect type, or be outside the condition defective type at least, the character of adenovirus carrier is not considered to the key point to Successful Practice of the present invention.Described adenovirus can be any of 42 kinds of known serotypes of difference or A-F subgroup.Adenovirus 5 types of C subgroup are for obtaining to be used for the preferred original material of condition replication-defective adenoviral vector institute of the present invention.This is that biological chemistry that they are a large amount of and genetic information are known, and this adenovirus is used to use in most of constructions of adenovirus as carrier traditionally always because adenovirus 5 types are human adenovirus.

As mentioned above, typical carriers of the present invention be replication defective and will be not gland-containing virus E1 district.Therefore, be introduce to transform construction the most easily to site that described E1 encoding sequence has been removed.Yet the on position of described construction in adenoviral sequence is not crucial for the purpose of the present invention.The polynucleotide of coding goal gene also can be inserted into and substitute as E3 replacement vector that (1986) such as Karlsson are described in the E3 district that lacks or be or helper virus has been replenished the E4 district of E4 defective at synergid.

The cultivation of adenovirus and processing are that those skilled in the art know, and present wide host range in vitro and in vivo.This papova can high titre obtain for example every milliliter 10 ⁹-10 ¹¹Plaque forming unit, and they are highly infectives.Do not need to be integrated into the host cell gene group life history of adenovirus.Foreign gene by the adenovirus carrier transmission is free and therefore host cell is had low genetoxic.The report that in research, still has no side effect (Couch etc., 1963 with the wild-type adenovirus immunization; Top etc., 1971), prove their security and treatment potentiality as vivo gene transmission carrier.

Adenovirus carrier has been used to expression (Levrero etc., 1991 of eukaryotic gene; Gomez-Foix etc., 1992) (Grunhaus and Horwitz, 1992 and in the vaccine development; Graham and Prevec, 1992).Recently, zooscopy prompting recombinant adenovirus can be used for gene therapy (Stratford-Perricaudet and Perricardet, 1991; Straford-Perricardet etc., 1990; Rich etc., 1993).The administered recombinant adenovirus comprises tracheal instillation (Rosenfeld etc., 1991 to the research of different tissues; Rosenfeld etc., 1992) intramuscular injection (Ragot etc., 1993), injection (Herz and Gerard, 1993) and directional inoculation are to brain (Le Gal La Salle etc., 1993) in the peripheral vein.

B.AAV infects

Adeno-associated virus (AAV) is to be used for attractive carrier of the present invention, because it has high-frequency integration and can infect nondividing cell, therefore make it enter mammalian cell useful (Muzyczka, 1992) in the tissue culture to transmitting gene.AAV has broad infection host scope (Tratschin etc., 1984; Larghlin waits 1986; Lebkowski etc., 1988; McLaughlin etc., 1988), this means that it can be used for purposes of the present invention.About produce and use the rAAV carrier be described in detail in U.S. Patent number 5,139,941 and U.S. Patent number 4,797,368 in description is arranged, this paper quotes each literary composition and is reference.

Proof uses the research of AAV to comprise (1988) such as LaFace in the gene transmission; Zhou etc. (1993); Flotte etc. (1993); With (1994) such as Walsh.Reorganization AAV carrier successfully has been used for external and transduction (Kaplitt etc., 1994 body internal labeling gene; Lebkowski etc., 1988; Samulski etc., 1989; Shelling and Smith, 1994; Yoder etc., 1994; Zhou etc., 1994; Hermonat and Muzyczka, 1984; Tratschin etc., 1985; McLaughlin etc., 1988), with the transduction (Flotte etc., 1992 that are used for the gene relevant with people's disease; Luo etc., 1994; 0hi etc., 1990; Walsh etc., 1994; Wei etc., 1994).Recently, the modified I phase people clinical experiment that is used for the treatment of cystic fibrosis of AAV carrier.

AAV is the dependency parvovirus because it need with other virus (member of adenovirus or simplexvirus family) cotransfection in culturing cell, to carry out productive infection (Muzyczka, 1992).Not with the helper virus cotransfection time, wild-type AAV genome is integrated into human 19 karyomit(e)s by its end, and it is with provirus form spend latent period (Kotin etc., 1990 there; Samulski etc., 1991).Yet, also express (Shelling and Smith, 1994) unless rAAV is not limited to the Rep albumen of the described AAV of 19 chromosomal integration.When carrying the proviral cell of AAV with the helper virus superingection, described AAV genome is from described karyomit(e) or from recombinant plasmid " recovery ", and sets up normal productive infection (Samulski etc., 1989; McLaughlin etc., 1988; Kotin etc., 1990; Muzyczka, 1992).

Generally speaking, make the viral method of reorganization AAV (rAAV) and comprise that cotransfection contains plasmid (McLaughlin etc., 1988 of the goal gene that has two terminal repetition flanking sequences of AAV; Samulski etc., 1989; This paper be cited as with reference to) and contain wild-type AAV encoding sequence and do not have the expression plasmid of terminal repeat, for example pIM45 (McCarty etc., 1991; This paper is cited as reference).Also available adenovirus or the plasmid that carries the adenoviral gene that needs the AAV subsidiary function of described cell infects or transfection.The rAAV virus original seed of making in this way contacts with adenovirus, described adenovirus must be with the rAAV particle physics on isolating (it is centrifugal for example to pass through cesium chloride density).Perhaps, can use the adenovirus carrier that contains the AAV coding region or contain clone (Yang etc., the 1994a of AAV coding region and some or all adenovirus auxiliary genes; Clark etc., 1995).Also can use and carry described rAAV DNA as being integrated proviral clone (Flotte etc., 1995).

C. retroviral infection

Retrovirus is a group single strand RNA virus, it is characterized by to change its RNA one-tenth double-stranded DNA (Coffin, 1990) by reverse transcription in infected cell.Then, the DNA stable integration of generation enters cell chromosome and and guides virus protein synthetic as provirus.Described integration causes described virus gene sequence to be retained in described recipient cell and the filial generation thereof.Described reverse transcription virus gene group contains three genes, gag, pol and env, their encode respectively housing albumen, polysaccharase and coating compositions.Described gag upstream region of gene finds have a sequence to contain the signal that the packaging gene group enters virion.Two long terminal repetition (LTR) sequences are present in described virus genomic 5 ' and 3 ' end.These genes contain strong promoter and enhancement sequences and also are to be incorporated into necessary in the described host cell gene group (Coffin, 1990).

For making up retroviral vector, the genetically modified nucleic acid of coding purpose is inserted into and replaces some virus sequence in the viral genome to produce replication defective virus.Be to produce virion, make up and contain gag, pol and env gene but do not contain described LTR and the package cell line (Mann etc. 1983) of packing composition.When the recombinant plasmid that contains cDNA is introduced in this clone with described retrovirus LTR and packing (for example by the calcium phosphate precipitation method), described packaging sequence makes this packaged virus particle that enters of rna transcription of described recombinant plasmid, entered substratum (Nicolas and Rubenstein, 1988 by secretion then; Temin, 1986; Mann etc., 1983).Then, collect the substratum contain described recombinant retrovirus, can choose wantonly concentratedly, and be used for transgenosis.Retroviral vector can infect the cellular type of wide variety.Yet, integrate the division (Paskind etc., 1975) that needs host cell with stably express.

Wild-type that relevant with the retroviral use of defective type is is duplicated competence virus and may be embodied in described packing cell.This can be caused by recombination event, wherein inserts the gag that is incorporated in the described host cell gene group, pol, env sequence upstream from the complete sequence of described recombinant virus.Yet, possibility (Markowitz etc., 1988 that present new used pack clone should reduce reorganization greatly; Hersdorffer etc. 1990).

D. other virus vector

Other virus vector can be used as construction of the present invention.Can use viral deutero-carrier such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etc., 1988) and simplexvirus deutero-carrier.They provide several attractive feature (Friedmann, 1989 for various mammalian cells; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etc., 1988; Horwich etc., 1990).

To with regard to the understanding of defective type hepatitis B virus, the new knowledge that is obtained is deep into the structure-functional relationship of different virus sequence with regard in the recent period.In vitro study shows that described virus can keep auxiliary dependency packing and reverse transcription ability, although disappearance is up to its genomic 80% (Horwich etc., 1990).This points out described genomic major part to be replaced by exogenous genetic material.Chang etc. introduce chloramphenicol acetyl transferasegene (CAT) recently and enter dhbv dna genome replacement polysaccharase, surface and front surface encoding sequence.It and wild-type virus cotransfection enter bird hepatoma cells system.The substratum that contains high titre recombinant virus is used to infector for young duck liver cell.Detect stable CAT genetic expression and after transfection, continue 24 days (Chang etc. 1991) at least.

In the further embodiment of other the present invention, it is a kind of by in the infective virus of through engineering approaches with expression specific combination part to think that described nucleic acid is encased in.Therefore, described virus particle is attached to the associated receptor of described target cell with specificity and transmits described composition to described cell.For the novel method that retroviral vector guiding become may design recently developed to the basis of the chemical modification method of described peplos by chemical method interpolation lactose residue.This modification method can make by the sialoglycoprotein acceptor carries out hepatocellular specific infection.

The method of another kind of recombinant retrovirus guiding is designed out, has wherein used at retroviral envelope protein matter with at the biotinylated antibody of specific cell acceptor.Described antibody is by using streptavidin and vitamin H composition coupling (Roux etc., 1989).Use the anti-main I of histocompatibility complex class and the antigenic antibody of II class, proved the various human cells (Roux etc., 1989) that carry these surface antigens of external use parent's preferendum virus infection.

4. coprecipitation of calcium phosphate or DEAE-dextran facture

In other preferred embodiment of the present invention, make described transgenic constructs be incorporated into described cell with the coprecipitation of calcium phosphate method.With the big T antigen transfection of SV40 the mouse primordial germ cells, result very good (Watanabe etc., 1997).Use this technology, people KB cell had been used adenovirus 5DNA transfection (Graham and Van Der Eb, 1973) already.Still by this way, mouse L (A9), mouse C127, CHO, CV-1, BHK, NTH3T3 and HeLa cell neomycin marker gene (Chen and Okayama, 1987) carried out transfection, and rat hepatocytes has carried out transfection (Rippe etc., 1990) with various marker gene.

In another embodiment, use the DEAE-dextran, make described expression constructs transmission enter described cell with polyoxyethylene glycol then, in this way, the report plasmid is introduced into mouse myeloma and erythroleukemia cell (Gopal, 1985).

5. directly microinjection or ultrasonic loading

Further embodiment of the present invention comprises by direct microinjection or ultrasonic loading introduces described transgenosis construct.Directly microinjection has been used to introduce nucleic acid construct thing (Harland and Weintraub, 1985) in the Africa xenopus ovum, and the LTK inoblast has carried out transfection (Fechheimer etc., 1987) with thymidine kinase gene by ultrasonic loading.

6. liposome-mediated conversion

In further embodiment of the present invention, described transgenosis construct can be coated in the liposome.Liposome is to be the cystic structures of feature with phospholipid bilayer film and internal water matrix.Multilamellar liposome contains a plurality of lipid layers that separated by water-based.They form when phosphatide is suspended in the excessive water solution immediately.Before forming imporosity, described lipid composition process is from rearrangement and surround water, and dissolved solute (Ghosh and Bachhawat, 1991) between described lipid layer.Desired also has a kind of and Lipofectamine (Gibco BRL) compound transgenosis construct.

The liposome-mediated nucleic acid transmission and the expression of external foreign DNA very successful already (Nicolau and Sene, 1982; Fraley etc., 1979; Nicolau etc., 1987).Wang etc. (1980) have proved in cultured chick embryo, in HeLa and the liver cancer cell, and the liposome-mediated transmission of foreign DNA and the feasibility of expression.

In certain embodiments of the invention, described liposome can form mixture with haemagglutinating virus (HVJ).This has shown is convenient to merge with described cytolemma and promoted the liposome of cell to catch enter (Kaneda etc., 1989) of DNA.In other embodiments, described liposome is can be with nuclear NHC protein (HMG-1) compound or unite use (Kato etc., 1991).In other further embodiment, described liposome is can be with HVJ and HMG-1 compound or unite use.

7. the relevant transfection of adenovirus

In certain embodiments of the invention, described transgenosis construct is incorporated in the described cell with the relevant transfection of adenovirus.The transfection efficiency that increases is existing (Kelleher and Vos, 1994 reported in using adenovirus link coupled cell system; Cotten etc., 1992; Curiel, 1994), and the present inventor expects to use same technology to increase transfection efficiency.

8. receptor-mediated transfection

Can be used for transmitting the further construction that described transgenosis construct enters described target cell is receptor-mediated transmission carrier.These carriers have the receptor-mediated endocytosis selectivity by occurring in target cell to absorb macromolecular advantage.According to the cellular type distribution of specific of various acceptors, this transmission method has increased degrees of specificity of the present invention.Wu and Wu (1993; This paper be cited as with reference to) the specificity transmission in the related content of another mammalian cell type described.

Some transgenosis is transmitted construction and is comprised cell receptor ligands specific and DNA binding reagents.Other comprises that DNA construction to be passed effectively has been attached to the cell receptor ligands specific on it.Several parts have been used to receptor-mediated transgenosis (Wu and Wu, 1987; Wagner etc., 1990; Ferkol etc., 1993; Perales etc., 1994; Myers, EPO0273085), it has set up the operability of described technology.

In other embodiments, described DNA transmits carrier components and can comprise the specific binding ligand relevant with liposome.Nucleic acid to be passed is packaged in the liposome, and described specific binding ligand combines with described liposome membrane on function.Therefore, described liposome specificity is attached to receptor in target cell and transmits described composition and give described cell.This type systematic has shown function when using following system, for example be used to receptor-mediated nucleic acid is passed to the cell that presents described EGF acceptor rise in described system's mesocuticle somatomedin (EGF).

Also have in the further embodiment, it can be liposome itself that the DNA of described transmission carrier transmits carrier components, and this will preferably include one or more and instruct cell-specific bonded lipid or glycoprotein.For example Nicolau etc. (1987) utilizes lactosyl ceramides porcine (a kind of asialo ganglioside of semi-lactosi end) to be incorporated into liposome and observes the increase of liver cell picked-up insulin gene.Expect that transgenosis construct of the present invention can be entered described target cell by the specificity transmission in a similar manner.

B. vector construction

The carrier that uses among the present invention contains the encoding sequence of a chosen gene of coding at least.Selected transgenosis can be the proteinic marker gene of coded markings, or another kind of purpose transgenosis (referring to following D sections and E sections).In addition, preferably contain marker gene and one or more additional genetically modified carriers.Described transgenosis preferably effectively puts together to drive described genetically modified transcribing with promotor.Enhanser can be included in the described carrier and transcribe with further enhancing.

The further embodiment of the carrier that uses among the present invention comprises and is positioned at described transgenosis flank and promotes described transgenosis homologous recombination to enter the genomic sequence of host animal.In certain embodiments, described carrier will comprise a cell transformation construction (for example oncogene), and it will make described clone immortalization to allow complicated genetic manipulation.In these embodiments, the transgenosis of described conversion will be positioned at and allow that this transgenosis under the felicity condition carries out the flank of the sequence of montage.By removing described conversion construction, described cell can be got back to its standard state, and is used to produce transgenic animal.In other embodiments, when needs finally removed described transgenosis from described host animal genome, the sequence that promotes transgenosis to excise can transform transgenosis with acellular and use.

C. promotor

The promotor and the enhanser of the genetic transcription of coded protein comprise a plurality of genes in the control eukaryotic cell.The adjusting information that each factor transmits can be assembled and integrate to described cell mechanism, allows that the different genes execution is distinctive, normally Fu Za transcriptional regulatory pattern.

The term promotor refers to that is transcribed a control unit as used herein, and its bunch collection is around the rna plymerase ii initiation site.The theory how most relevant promotors are organized comprises HSV thymidine kinase (tk) and the SV40 promoter Analysis of transcription unit early from the analysis to several viral promotors.These researchs (being enriched by more recent work) have shown that promotor comprises discrete functional module, and each assembly is made up of the DNA of about 7-20bp, and comprises the cog region of one or more transcription activating albumen.

In each promotor, have at least an assembly to play the function of the synthetic initiation site of location RNA.The example of wherein knowing the most is the TATA box, but in some promotor, there is not the TATA box, as the promotor of mammiferous terminal deoxyribonucleotidyl transferase gene and the promotor of SV40 gene in evening, the discrete factor self that is overlapped in initiation site is assisted fixedly zero position.

The other promotor factor is regulated the transcription initiation frequency.Generally speaking, these factors are positioned at the zone of the 30-100bp of initiation site upstream, although many promotors had demonstrated the functional factor that also contains the initiation site downstream recently already.Space between the factor is variable so that reversing or when mobile promotor still keep function.In the tk promotor, the space between the factor can be added to 50bp at interval before activity begins to descend.As if according to the difference of promotor, each factor can play the function of collaborative or independent activated transcription.

Enhanser is to be detected as the gene that enhancing is positioned at non-conterminous locational promoter transcription on the same DNA molecule at first.This is rare precedent in the protokaryon transcriptional regulatory research of ability at classics that remote span works.The active DNA of the enhanser district that studies show that subsequently is organized to such an extent that resemble very much promotor.In other words, they comprise many independent factors, wherein each factor and one or more protein bound of transcribing.

Basic difference between enhanser and the promotor is to use.The enhancing subarea must stimulate to be positioned at a distance as an integral body transcribes; This is not real needs for promotor or its composing factor.On the other hand, promotor must have one or more to instruct at particular location and with the initial RNA synthetic of the concrete direction factor, and enhanser does not have these characteristics.Except the difference that this work is used, enhanser and promotor are very similar entities.

Promotor and enhanser have the basic function of transcribing in the same activating cells.They are eclipsed and successive normally, looks usually to form mode by very similar assembly.In a word, these aspects prompting enhansers and promotor are with source entity and are attached to transcription activating protein matter the same manner and the machine-processed interaction of cell transcription substantially on these sequences.

In anything part, will be understood that promotor is the DNA factor, it causes this expression of gene when functionally residing in the upstream of a gene.Be positioned at the downstream of promoter element on most transgenosis construct function of the present invention.

1. the promotor special to undifferentiated cell

What preferably use in the present invention is promoters active in undifferentiated cell.Research had shown already that for CMV promotor for expressing in the undifferentiated cell be not (embodiment 3) optimized.Cause that the promotor of high level expression comprises that phosphoglyceric kinase (pgk) promotor and eight aggressiveness are in conjunction with transcription factor 4 (Oct-4) promotor in undifferentiated cell.The known high level expression that causes in not breaking up mouse ES cells of Pgk promotor.

Transgenic experiments has been identified Oct-4 upstream region of gene regulatory region, and it can play beginning and end differentiation inner cell matter and genitaloid high level expression (Yeom etc., 1996).By using the Oct-4 promotor, expression will be limited in highly undifferentiated cell and PGCs, allow that thus the expression level by measuring screened labelled protein (preferred egfp) has the cell of the effect of high probability with screening to described germ cell line.Use the Oct-4 promotor to consider that also the transgenic animal to inferring carry out early screening, because of reaching that test does not need to preserve mosaic before with reproduction age and because of not needing costliness and tediously long breeding to test plenty of time and the expense of saving.

Present inventor's expectation is from various animal species clone Oct-4 genes (seeing the V joint), for using in the present invention.

Eucaryon with virus promotor and enhanser

Preferred use is cytomegalovirus (CMV) promotor among the present invention.This promotor can buy with the form the carrier pcDNAIII from Invitrogen company, and described product preferably uses in the present invention.Be other viral promotors below, the tabulation of cell promotor/enhanser and inducible promoters/enhanser, they can be used in combination with the present invention.In addition, any promotor/enhanser combination (according to eukaryotic promoter database EPDB) can be used to drive coding oligosaccharides processive enzyme, protein folding accessory protein, but the expression of the structure gene of selection markers protein or purpose heterologous protein.

Table 1

But inducible factor factor inductor reference MTII Buddhist ripple ester (TFA) Palmiter etc., 1982; Haslinger and

Heavy metal Karin, 1985; Searle etc., 1985;

Stuart etc., 1985; Imagawa etc.,

1987; Karin ^, 1987; Angel etc.,

1987b; McNeall etc., 1989MMTV (mouse mammary tumour virus) glucocorticosteroid Huang etc., 1981; Lee etc., 1981;

Majors and Varmus, 1983; Chandler

Deng, 1983; Lee etc., 1984; Fonta

Deng, 1985; Sakai etc., poly-(rI) X Tavernier of 1986 interferon-etc., 1983

Poly-(rc) adenovirus 5E2 Ela Imperiale and Nevins, 1984 collagenase Buddhist ripple ester Angle etc., 1987a stromelysin Buddhist ripple ester Angle etc., 1987bSV40 Buddhist ripple ester Angle etc., 1987b mouse MX gene Interferon, rabbit, Avian pneumo-encephalitis virus GR78 gene A 23187 Resendez etc., 1988 α-2-macroglobulin IL-6 Kunz etc., 1989 vimentin serum Rittling etc., 1989MHC I genoid H-2 κ b Interferon, rabbit Blanar etc., 1989HSP70 Ela, SV40 large T antigen Taylor etc., 1989; Taylor and

Kingston, 1990a, b proliferin Buddhist ripple ester-TPA Mordacq and Linzer, 1989 tumour necrosis factor FMA Hensel etc., 1989 thyroxine yield stimulant α gene Triiodothyronine Chatterjee etc., 1989

Table 2

Other promotor/enhanser factor promotor/enhanser reference heavy chain immunoglobulin Hanerji etc., 1983; Gilles etc., 1983; Grosschedl and

Baltimore, 1985; Atchinson and

Perry, 1986,1987; Imler etc., 1987; Weinberger etc.,

1988; Kiledjian etc., 1988; Porton etc., 1990 light chain immunoglobulin Queen and Baltimore, 1983; Picard and Schaffner 1984T cell receptor Luria etc., 1987; Winoto and Baltimore, 1989 Redondo

Deng, 1990HLA DQ and DQ β Sullivan and Peterlin, 1987 Goodbourn etc., 1986; Interferon-Fujita etc., 1987 Goodbourn and Maniatis, 1985 interleukin II Green etc., 1989 interleukin 2 receptor Green etc., 1989; Lin etc., 1990MHCII class 5 Koch etc., 1989MHC II class HLA-DR α Sherman etc., 1989 beta-actin Kawamoto etc., 1988; Ng etc., 1989 muscle creatine kinase Jaynes etc., 1988; Horlick and Benfield, 1989;

Johnson etc., 1989a prealbumin (fortune thyroxine Costa etc., 1988 albumen) elastoser I Omitz etc., 1987 metallothionein(MT) Karin etc., 1987; Culotta and Hamer, 1989 collagenase P inkert etc., 1987; Angel etc., 1987 globulin gene Pinkert etc., 1987; Tronche etc., 1989,1990 alpha-fetoprotein Godbout etc., 1988; Campere and Tilghman, 1989t-globin Bodine and ley, 1987; Perez-Stable and Constantini,

1990 beta globin Trudel and Constantini, 1987e-fos Cohen etc., 1987c-HA-ras Triesman, 1986; Deschamps etc., 1985

Table 2 (continuing) promotor/enhanser reference Regular Insulin Edlund etc., 1985 neurocyte attachment molecules (NCAM) Hirsch etc., 1990al-antitrypsin Latimer etc., 1990H2B (TH2B) histone Hwang etc., 1989 mouse or type i collagen Ripe etc., 1989 glucose-adjusting PROTEIN C hang etc., 1989 (GRP94 and GRP78) rat growth hormone Larsen etc., 1986 human serum amyloid A (SAA) Edbrooke etc., 1989 Troponin Is (TNI) Yutzey etc., 1989 Thr6 PDGF BB Pech etc., 1989 duchenne muscular dystrophy Klamut etc., 1990SV40 Baner ji etc., 1981; Moreau etc., 1981; Sleigh and Lockett, 1985; Firak

And Subramanian, 1986; Herr and Clarke, 1986; Imbra and

Karin, 1986; Kadesch and Berg, 1986; Wang and Calame, 1986; Ondek etc.,

1987; Kuhl etc., 1988 polyoma Swartzendruber and Lehman such as 1987 Schaffner, 1975; Vasseur etc., 1980; Katinka

Deng, 1980,1981; Tyndell etc., 1981; Dandolo etc. 1983; De

Villiers etc., 1984; Hen etc., 1986; Satake etc., 1988;

Campbell and Villarreal, 1988 retrovirus Kriegler and Botchan, 1982,1983; Levinson etc., 1982;

Kriegler etc., 1983,1984a, b, 1988; Bosze etc., 1986; Miksicek

Deng, 1986; Celander and Haseltine, 1987; Thiesen etc., 1988;

Celander etc., 1988; Chol etc., 1988; Reisman and Rotter, 1989 papillomavirus Campo etc., 1983; Lusky etc., 1983; Spandidos and

Wilkie, 1983; Spalholz etc., 1985; Lusky and

Botchan, 1986; Cripe etc., 1987; Gloss etc., 1987; Hirochika

Deng, 1987, Stephens and Hentschel, 1987; Glue etc., 1988

Table 2 (continuing) promotor/enhanser reference hepatitis B virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986; Sbaul

And Ben-Levy, 1987; Spandau and Lee, 1988; Vannice and

Levinson, 1988 human immunodeficiency virus Muesing etc., 1987; Hauber and Cullan, 1988; Jakobovits

Deng, 1988; Feng and Holland, 1988; Takebe etc., 1988;

Rowen etc., 1988; Berkhout etc., 1989; Laspia etc.,

1989; Sharp and Marciniak, 1989; Braddock etc., 1989 cytomegalovirus Weber etc., 1984; Boshart etc., 1985; Foecking and

Hofstetter, 1986 gibbon ape leukemia virus Holbrook etc., 1987; Quinn etc., 1989

D. marker gene and protein

The present invention also provides candidate's recombinant screen and system of selection, described method is based on full cell detection and preferably utilize reporter gene, reporter gene is given its recon host and is easy to detected phenotype, and described phenotype only has under the condition of function at the common DNA that is positioned at this report upstream region of gene and just occurs.Generally speaking, reporter gene encoded polypeptides (labelled protein) is no longer produced in addition by this host cell, and it can detect by analyzing described cell culture, and for example by fluorometric assay, radio isotope or spectrophotometer are analyzed described cell culture.

1. screening

The enzyme that exemplifies comprises esterase, Phosphoric acid esterase, and proteolytic enzyme (tissue plasminogen activator or urokinase) and other can be by its active enzymes that is detected, as known to those skilled in the art.The more preferably egfp (GFP) of Shi Yonging is as the marker (Chalfie etc., 1994) of transgene expression in the present invention.The substrate that the use of GFP does not need external source to add only passes through closely ultraviolet ray or blue light illumination, thereby the potentiality of using in the genetic expression in the monitoring viable cell are obvious.The correct evaluation by the previous system of selection that just has of modification cell need be cultivated in being called the chemical reagent of G418 processed cell 10-14 days, was necessary between the system of selection effective date cell transfer in new feeder cell.Yet egfp (GFP) makes as a kind of use of differentiating mark and can identify the transgenic cell colony and need not go down to posterity or bring Selection In substratum.As a result, described cell keeps more healthy and has kept it to move or blastocyst injection back produces the offspring's that lives ability at consideration convey owing to do not go down to posterity repeatedly.

Other preferred embodiment is E.C. 2.3.1.28 (CAT), it can with radio-labeled substrate, Lampyridea and bacterial luciferase, and bacterium beta galactosidase enzyme and β glucuronidase use together.Marker gene in other this type of is well known to those skilled in the art, and is suitable for using in the present invention.

2. select

Another kind of but the reporter gene of detected characteristics is provided on host cell is coded polypeptide, gives the mark of the general enzyme of its transformant toxinicide resistance.The example of this type of reporter gene is neo gene (Colberre-Garapin etc., 1981), and its protection host cell is resisted the toxic level of microbiotic G418, gene (the United States Patent (USP) 4 of streptomycin resistance is provided, 430,434) (Santerre etc. 1984, to give the gene of hygromycin B resistance; United States Patent (USP) 4,727,028,4,960,704 and 4,559,302), the gene (it gives the resistance to methotrexate) (Alt etc., 1978) of a coding Tetrahydrofolate dehydrogenase, gene and many other genes well-known in the art (Kaufman, 1990) of coding HPRT.

E. purpose transgenosis

As mentioned above, transgenic animal have been widely used.Its applicability realizes that by introducing different types of gene the kind that is introduced into gene depends on needed target.Following table 3 and table 4 are to be incorporated into some limiting examples in the transgenosis type of non-rodent by the inventive method.

The former disease antigen reference of table 3 oral immunity amebic dysentery SREHP Zhang etc., 1995 infections with leishmaniasis GP63 Xu etc., 1995 respiratory tract infection (pig) pleuropneumonia unwrapping wire bar Hensel etc., 1995

Bacterium Jain and Michael, 1995 respiratory tract infection (pig) Pseudomonas aeruginosa

OprF Cripps etc., 1995 hepatitis autoimmune disease multiple sclerosis myelin basic protein matter Weiner etc., 1995 diabetes (autoimmune disease type) Regular Insulin Weiner etc., 1995

Table 3 (continuing) production of antibodies is transplanted the protein reference

HCD59 Kroshus etc., 1996

DAF (CD55) van Denderen etc., 1996

The proteinic modified biological medicine of I type and II type MHC is transplanted protein reference christmas factor IX Clark etc., 1989

Urokinase Meade etc., 1990 wind-puff alpha-1 antitrypsin Archibald etc., 1990 Gordon of apoplexy tissue plasminogen activator etc., 1987 cancer interleukin-Buhler etc., 1989 burn collagen cancer Interferon, rabbit Houdebine, 1994 heart trouble C albumen Houdebine, 1994 growth Pursel and Rexroad

1993 disease resistance Brem, 1993; Clements

Deng, 1994; Lo etc.,

1991; Weidle etc., 1991 wood feature Bullock etc., 1995

Table 3 (continuing) milk-content (is selected from Clark, 1992; Yom and Bremel, 1993;

Houdebine 1994) the change result increases α and the β casein improves curdled milk hardness in order to cheese production

Promote thermostability, increase calcium contents and increase increase calcium contents in phosphorylation position in the casein, promote emulsification to select the speed (improving the cheese delicious degree) that introducing albumen water increase quality forms in casein and separate the stability that the site increases κ casein concentration increase casein polymkeric substance, reduce the micelle size

Reduce gelation and aggegation and eliminate beta lactoglobulin reduction high temperature gelization, improve digestibility, reduce irritated anti-

Should, reduce the elementary source of Ruzhong halfcystine and reduce alpha lactalbumin minimizing lactose, increase the market potential of fluid dairy products, reduce ice

Brilliant formation, the mammary gland osmoregulation that is in harmonious proportion add human milk iron transfer albumen enhancing iron and absorb, and prevent that visceralization from adding the proteolysis site to the κ casein

Increase the cheese curing speed and reduce acetyl-CoA carboxylase reduction lipid content, improve nutritional quality, reduce dairy production

The Equivalent that cost is expressed immunoglobulin gene defence cause of disease such as Salmonellas and Listera personnel selection replaces cow's milk protein plasmagene anthropomorphic dummy breast

Table 3 (continuing) other protein-based each member's hematoglobin protein Rh factor VIII and IX, complement factor or composition, oxyphorase hormone Regular Insulin, tethelin, Triiodothyronine, catecholamine, sugared skin

The matter hormone, PMSG, trop(h)ic hormone, prolactin antagonist, pitocin, Dopamine HCL somatomedin EGF, PDGF, NGF, IGF cytokine interleukin-, CSF, GMCSF, TNF-α, TGF-α and TGF

β enzyme tissue plasminogen activator, streptokinase, cholesterol biosynthesizing agent

Or degradation agents, digestive pharmaceutical, steroid generates agent, kinases, di(2-ethylhexyl)phosphate

Esterase, methylase, demethylation enzyme, desaturase, Mierocrystalline cellulose

Enzyme, proteolytic enzyme, glycosylase, lipase, phospholipase, aromatize

Enzyme, cytopigment, adenylic acid (AMP) or guanylate cyclase hormone or other acceptor LDL, HDL, steroid, protein, peptide, the conjugated protein steroid of fat or prostaglandin(PG), tethelin or growth factor binding proteins immune system protein matter antibody, SLA or mhc gene antigen bacterial antigens, parasite antigen, virus antigen, sensitinogen muscle protein myosin, tropomyosin

Table 4

Selected clone's structure gene gene clone type ^*Reference activin pig-cDNA Mason AJ, Nat, 318:659,1985 adenosine deaminase h-cDNA Wiginton DA, PNAS, 80:7481,1983 angiotensinogen I r-cDNA Ohkubo H, PNAS, 80:2196,1983

R-gDNA Tanaka T, JBC, 259:8063,1984 Antithrombin III H-cDNA Bock SC, NAR 10:8113,1982

H-cDNA and gDNA Prochownik EV, JBC, 258:8389,

1983 alpha1 Anti-trypsin h-cDNA KurachiK, PNAS, 78:6826,1981

h-gDNA????????????Leicht?M，Nat，297：655，1982

RFLP Cox DW, AJHG, 36:134S, 1984 apoCs-1 h-cDNA Knott TJ, NAR, 12:3909,1984 apoCs-II h-cDNA Jackson CL, PNAS, 81:2945,1984

h-cDNA????????????Mykelbost?O，JBC，249：4401，1984

h-cDNA????????????Fojo?SS，PNAS，81：6354，1984

RFLP??????????????Humphries?SE，C?Gen，26：389，

1984 apoCs-III h-cDNA and gDNA Karanthanasis SK, Nat, 304:371,

1983

H-cDNA Sharpe CR, NAR, 12:3917,1984 apo E h-cDNA Brewslow JL, JBC, 257:14639,

1982 atrial natriuretic peptide h-cDNA Oikawa S, Nat, 309:724,1984

h-cDNA????????????Nakayama?K，Nat，310：699，1984

h-cDNA????????????Zivin?RA，PNAS?81：6325，1984

h-gDNA????????????Seidman?CE，Sci，226：1206，1984

h-gDNA????????????Nemer?M，Nat，312：654，1984

H-gDNA Greenberg BI, Nat, 312:665,1984 chorionic-gonadotropin hormone h-cDNA Fiddes JC, Nat, 281:351,1981 α chain RFLP Boethby M, JBC, 256:5121,1981

Table 4 (continuing)

Selecteed cloning structure gene gene clone type ^*Reference rennin ox-cDNA Harris TJR, NAR, 10:2177,1982 complements, B factor h-cDNA Woods DE, PNAS, 79:5661,1982

H-cDNA and gDNA Duncan R, PNAS, 80:4464,1983 complement C2 h-cDNA Bentley DR, PNAS, 81:1212,1984

h-gDNA(C2，C4???????Carroll?MC，Nat，307：237，1984

With B complement C3 m-cDNA Domdey H, PNAS, 79:7619,1983

H-gDNA Whitehead AS, PNAS, 79:5021,1982 complement C4 h-cDNA and Gdna Carroll MC, PNAS, 80:264,1983

H-cDNA Whitehead AS, PNSA, 80:5387,1983 Urogastron m-cDNA Gray A, Nat, 303:722,1983

m-cDNA??????????????Scott?J，Sci，21：236，1983

H-gDNA Brissenden JE, Nat, 310:781,1984 Urogastron h-cDNA and Chr Lan CR.Sci, 224:843,1984 oncogene c-erbB acceptor epoxide dehydration r-cDNA Gonzlalez FJ, JBC, 256:4697,1981 enzyme erythropoietin h-cDNA Lee-Huang S, PNAS, 81:2708,1984Cl esterase suppress h-cDNA Stanley KK, EMBO J, 3:1429,1984 doses, Cl Factor IX h-cDNA and gDNA Gitschier J, Nat, 312:326,1984

H-cDNA Toole JJ, Nat, 312,342,1984 factors IX, gram h-cDNA Kutachi K, PNAS, 79:6461,1982 li this Maas factor h-cDNA Choo KH, Nat, 299:178,1982

RFLP????????????????Camerino?G，PNAS，81：498，1984

h-gDNA??????????????Anson?DS，EMBO?J，3：1053，1984

Table 4 (continuing)

Selecteed cloning structure gene gene clone type ^*Reference B beta, gamma h-gDNA (γ) Fornace AJ, Sci, 224:161,1984

h-cDNA(αγ)????????Imam?AMA，NAR，11：7427，1983

H-gDNA (γ) Fornace AJ, JBC, 259:12826,1984 gastrin releasing peptide h-cDNA Spindel ER, PNAS, 81:5699, hyperglycemic-glycogenolytic factor hamster c-DNA Bell GI before 1984, Nat, 302:716,1983 is former

H-gDNA Bell GI, Nat, 304:368,1983 tethelin h-cDNA Martial JA, Sci, 205:602,1979

h-gDNA??????????????DeNoto?FM，NAR，9：3719，1981

GH sample gene Owerbach, D, Sci, 209:289,1980 tethelin, h-cDNA Gubler V, PNAS, 80:3411,1983RF, supressor pig-cDNA Mason AJ, Nat, 318:659,1985 preproinsulin h-gDNA Ullrich a, Sci, 209:612,1980 insulin-like growth h-cDNA Jansen M, Nat, 306:609,1983 factor I

h-cDNA??????????????Bell?GI，Nat，310，775，1984

Chr Brissenden JE, Nat, 310,781,1984 insulin-like growth h-cDNA Bell GI, Nat, 310:775,1984 factor II

h-gDNA??????????????Dull?TJ，Nat，310：777，1984

Chr?????????????????Brissenden?JE，Nat，310：781，1984

Table 4 (continuing)

Selecteed cloning structure gene gene clone type ^*Reference interferon-alpha h-cDNA Maeda S, PNAS, 77:7010,1980 (white corpuscles), multiple h-cDNA (8 kinds) Goeddel DV, nat, 290:20,1981

h-gDNA??????????????Lawn?RM，PNAS，78：5435，1981

h-gDNA??????????????Todokoro?K，EMBO?J，3：1809，1984

H-gDNA Torczynski RM, PNAS, 81:6451,1984 interferon-h-cDNA Taniguchi T, Gene, 10:11,1980 (inoblast) h-gDNA Lawn RM, NAR, 9:1045,1981

H-gDNA (relevant) Sehgal P, PNAS, 80:3632,1983

H-gDNA (relevant) Sagar AD, Sci, 223:1312,1984 interleukin 1 m-cDNA Lomedico PT, Nat, 312:458,1984 interleukin IIs, T cell h-cDNA Devos R, NAR, 11:4307,1983 somatomedin h-cDNA Taniguchi T, Nat, 302:305,1983

h-gDNA??????????????Hollbrook?NJ，PNAS，81：1634，1984

Chr Siegel LF, Sci, 223:175,1984 interleukin 3 m-cDNA Fung MC, Nat, 307:233,1984 prokinins, two kinds of configuration ox-cDNA Nawa H, PNAS, 80:90,1983

Ox-cDNA and gDNA Kitamura N, Nat, 305,545,1983 interstitialcellstimulating hormone (ICSH)s, h-gDNA of β subunit and Chr Talmadge K, Nat, 207:37,1984 lymphotoxin h-cDNA and gDNA Gray Pw, Nat, 312:721,1984 mast cell growth factor m-cDNA Yokoya T, PNAS, 81:1070,1984 nerve growth factors, the m-cDNA Scott J of β subunit, Nat, 302:538,1983

h-gDNA??????????????Ullrich?A，Nat，303，821，1983

Chr Franke C, Sci, 222:1248,1983 oncogenes, c-sis, PGDF h-gDNA Dalla-Favera R, Nat, 295:31,1981A chain h-cDNA Clarke MF, Nat, 208:464,1984 pancreas polypeptide and twenty peptide h-cDNA Boel E, EMBO J, 3:909,1984 Pre Pro PTH h-cDNA Hendy GN, PNAS, 78:7365,1981

h-gDNA??????????????Vasicek?TJ，PNAS，80：2127，1983

Table 4 (continuing)

Selecteed cloning structure gene gene clone type ^*Reference profibr(in)olysin h-cDNA and gDNA Malinowski DP, Fed P, 42:1761,

1983 profibr(in)olysins swash h-cDNA Edlund T, PNAS, 80:349,1983 beings

h-cDNA????????????????Pennica?D，Nat，301：214，1983

H-gDNA Ny T, PNAS, 81:5355,1984C albumen h-cDNA Foster D, PNAS, 81:4766,1984 zymoplasms aurochs-cDNA MacGillivray

RTA, PNAS, 77:5153,1980 Relaxin h-gDNA Hudson P, Nat, 301:628,1983

H-cDNA (2 gene) Hudson P, EMBO J, 3:2333,1984

Chr???????????????????Crawford，RJ，EMBO

J, 3:2341,1984 preprogastrin h-cDNA Imai T, PNAS, 80:7505,1983

h-gDNA????????????????Hobart?PM，PNAS?81：5026，1984

h-gDNA????????????????Miyazaki?H，PNAS，81：5999，1984

Chr Chirgwin JM, SCMG, 10:415,1984P﹠amp; K material ox-gDNA Nawa H, Nat, 312:729,1984 urokinase h-cDNA Verde P, PNAS, 81:4727, the 1984 preceding former h-cDNA Itoh of vasoactive intestinal peptide N, Nat, 304:547,1983 vassopressin r-cDNA Schmale H, EMBO J, 2:763,1983

Table 4 keyword: ^*The cDNA-complementary DNA; Chr-karyomit(e); The gDNA-genomic dna; The RFLP-restrictive fragment length polymerphism; H-people; The m-mouse; The r-rat

1. oral vaccine

This proteinoid has immunology and is worth when producing in other target organ Mammals body of gland or transgenic animal.These protein can be purified also in conjunction with the specific immunogens orally give that is used to produce oral vaccine then.Some candidate albumen matter of using among the present invention is hepatitis and rabies virus antigen.

2. oral tolerance

This type of is protein-based to be similar to top the sort of protein, and different is that this protein is by stimulating tolerance to replace the transgene mammal of the compound of immune stimulatory power to produce.Infer that this protein has the treatment autoimmune disease such as multiple sclerosis is sick and the potential use of diabetes.This type of preferred protein that uses among the present invention comprises the Regular Insulin of the myelin basic protein matter and the treatment diabetes of treatment multiple sclerosis disease.

3. bio-pharmaceuticals

This is meant in transgene mammal body of gland and other organ produces medical treatment and the animal doctor uses compound.This proteinoid can be used for treating the fire victim, cardiac, hemophilia and paralytic.Preferred this proteinoid of using among the present invention comprises alpha1 Anti-trypsin, collagenase, Factor IX, factors IX and tissue plasminogen activator.

4. transplant

The maximum potential use of homologous recombination technique in non-rodent is to produce the ubiquity donor animal.These animals can have the gene relevant with the tissue rejection inactivation, therefore make described tissue transplantation enter human body on interim basis.This has just greatly reduced the shortage phenomenon of present donor organ.The gene relevant with the repulsion process that uses among some suitable the present invention is hCD59, DAF (CD55) and I class and the II class MHC molecule modified.

5. animal model of human disease

By in the ES cell, using homologous recombination, the existing animal host that may create the medical condition that has the simulating human disease.The example of these models is atherosclerosiss, cystic fibrosis and Alzheimer disease.Regrettably, in some cases, mouse is not a kind of ideal animal model.The present invention allows to produce the macrofauna model of human diseases, and they can use with developing new drug in scientific circles and private business, and deepens the understanding to disease specific.For example, a purposes of the present invention is to produce apo E defective pig.The present inventor had proved before that apoE defective type pig produced spontaneous premature atherosclerosis (Piedrahita etc., 1992; Zhang etc., 1992).Close copy in the macrofauna (as pig) will be the great wealth of developing drugs (comprising gene therapy) with the treatment human diseases.

6. agronomic traits

At agriculture field, a large amount of uses of transgenic animal aspect are existing to be described, and their available methods of the present invention produce.Nonrestrictive a series of purposes comprises that generation is to some disease and the resistive animal of insect, the modified dairy moiety is to extend the shelf life, cheese yield and the dairy products that lactose intolerance receptor energy safe edible is modified, change the growth rate of animal, nutrient efficiency and meat composition, and as influencing the purposes of timber quality aspect.For example has something to do to the gene of dairy products improvement be α-, β-and κ-casein, lactoglobulin and whey-protein.Changing the preferred gene that uses in the meat is GDF-8 (McPherron etc., 1997).

Another proterties of using among the present invention is to produce the ox that does not have prion protein (PrP).Recent research has shown that the mouse that does not have PrP has resistibility (Bueler etc., 1992,1993 to scrapie virus; Brandner etc., 1996; Fischer etc., 1996; Blattler etc. 1997).Spongy encephalitis (BSE) media is considered to most of to be made up of (if not all) prPSc (the unusual isomer of normal cell PrPC), does not therefore have the ox of PrP resistibility to be arranged to BSE.The gene of coding ox PrP is known (Goldmann etc., 1991; Inoue etc., 1997).

7. antisense method

The antisense methodology is utilized nucleic acid and " complementation " sequence paired fact in opposite directions.Since complementary, mean that polynucleotide can carry out base pairing according to the complementary rule of the Watson-Crick of standard.In other words, bigger purine will carry out with less pyrimidine base pairing form guanine and cytosine(Cyt) (G: C) pairing, and when DNA VITAMIN B4 and thymus pyrimidine (the A: (A: U) match and combine of VITAMIN B4 and uridylic T) or during RNA.In hybridization sequences, comprise extremely uncommon base such as inosine, 5-methylcytosine, 6 methyladenines, xanthoglobulin and other base are not disturbed pairing.

Double-stranded (ds) DNA causes triple helix to form with the polynucleotide guiding; Guiding RNA will cause duplex to form.Antisense polynucleotides combines with their target polynucleotide specificity when being introduced into target cell and disturbs and transcribe, RNA processing, transhipment, translation and (or) stability.In the transgenic animal that produced by the primordial germ cells that transformed and the present invention, the sense-rna construction, or the DNA of this type of sense-rna s that encodes can be utilized to suppressor gene and transcribes or translate or both.

Can design promotor and other control region of antisense construct thing and selected genes, even exon is intron exon-intron boundary combination.Expect that effective antisense construct thing will generally include and intron (exon) fragment meet complementary zone.Therefore, with 50-200 base of the intron-exon fragment meet of a selected genes in the antisense construct thing of regional complementarity be supposed to be used for this method.Observed already that some exon sequence can be included in this construction and not seriously influenced its target selection.The amount of included exon will be different and different with intron sequences with the concrete exon that uses.Whether as long as be affected with other expression of gene that defines complementary sequence by these constructions of vitro detection just to detect the exon DNA that whether is comprised easily too big for the experimenter.

" antisense " or " complementation " means the complementation that covers its total length basically and the polynucleotide sequence that mispairing is seldom arranged.For example length is the sequence of 15 bases, when it is called complementation when there is complementary nucleotide 13 or 14 positions.Nature, fully the complementary sequence will be that its whole total length is complementary fully and do not have a sequence of any mispairing.Other low degree homologous sequence also is considered.For example, can design the antisense construct thing (for example ribozyme) that has limited height homology zone but also contain non-homogeneous zone.These molecules are considered to be lower than 50% homology, and they will combine with target sequence under suitable condition.

It may be favourable to producing concrete construct that portion gene group DNA is combined with cDNA or synthetic sequence.For example, in final construction, need the place of an intron, just need to use genomic clone.Described cDNA or synthetic polynucleotide can provide restriction site more easily with the described construction of retained part and therefore will be used to the rest part of described sequence.

8. ribozyme

Though protein is used to analysis of nucleic acids traditionally, has occurred another kind of macromole and used as this function.Ribozyme is the RNA-protein complex, and they cut nucleic acid in the site-specific nature mode.Ribozyme has single-minded catalysis region, and there are endonuclease enzymic activity (Kim and Cech, 1987 in described zone; Gerlach etc., 1987; Forster and Symons, 1987).For example a large amount of ribozymes quickens to have highly narrow spectrum phosphoester transfer, only cuts one of several phosphoric acid ester in the oligonucleotide substrate (Cech etc., 1981 usually; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).This species specificity is by owing to before the chemical reaction, and substrate is by internal guide sequence (" IGS ") the bonded needs of single-minded match reaction with described ribozyme.

A part (Joyce, 1989 that the ribozyme catalysis reaction is mainly the sequence relevant with nucleic acid-single-minded cutting (connecing) reaction have been observed; Cech etc., 1981).For example, U.S. Patent number 5,354,855 some ribozyme of report can play the narrow spectrum endonuclease of bigger sequence is arranged and near the specificity of DNA restriction enzyme than known rnase.Therefore the restraining effect to genetic expression of sequence specificity ribozyme mediation can be particularly suitable for therepic use (Scanlon etc., 1991:Sarver etc., 1990; Sioud etc., 1992).Reported the hereditary change that ribozyme causes in having used some clone of ribozyme recently; Reformed gene comprises oncogene H-ras, c-fos and HIV gene.Most of this respect researchs comprise that the single-minded sudden change codon according to single-minded ribozyme cutting carries out the modification of said target mrna.

Several different ribozyme primitives are described (Symons, 1992) with the RNA nicking activity.Be considered to be equivalent on the function low K _mThe example of the negative adjusting of hexokinase comprises the I group's of self montage intron sequence, comprises nepovirus (Prody etc., 1986), avocado sunspot viroid (Palukaitis etc., 1979 and Symons, 1981), with the instantaneous streak virus of clover (Forster and Symons, 1987).According to the desired secondary structure of folding up, the sequence of these and relevant viral source is called hammerhead ribozyme.

Other ribozyme that is fit to includes the sequence from RNase P (Yuan etc., 1992, the Yuan and the Altman of RNA nicking activity, 1994), hair clip ribozyme structure (Berzal-Herranz etc., 1992 and Chowrira etc., 1993) and based on the ribozyme of hepatitis D virus.The generality design of the RNA nicking activity that ribozyme instructs and optimizing has a detailed description (Haseloff and Gerlach, 1988, Symons, 1992, Chowrira etc., 1994 and Thompson etc., 1995).

Other variable quantity in the ribozyme design be the selection of the cleavage site on the given target RNA.Ribozyme is annealed to a site by complementary base to interaction and is directed to a given sequence.Two homology segments are that this guiding is needed.These homologous sequence sections are at the flank of the catalysis ribozyme structure of above-mentioned definition.Each section of homologous sequence can not wait by from 7 to 15 Nucleotide on length.The unique needs that define this homologous sequence are that the single-minded sequence that they are used as cleavage site on described target RNA separates.With regard to hammerhead ribozyme, cleavage site is a dinucleotide sequence on the target RNA, promptly connects VITAMIN B4 behind the uridylic (U), cytosine(Cyt) or uridylic (A, C or U) (Perriman etc., 1992 and Thompson etc., 1995).On the occurrence frequency statistics of this dinucleotide in any given RNA is 3/16ths.Therefore, with regard to the target messenger RNA(mRNA) of 1000 given bases, the cleavage site of 187 dinucleotides is possible on the statistics.

The target RNA of design and detection ribozyme effectively cutting is a method well-known in the art.The example of the scientific approach of design and detection ribozyme is by (1994) and Lieber and Strauss (1995) descriptions (each literary composition is cited as reference) such as Chowrira.In the ribozyme of selected genes guiding, use effectively and the evaluation of preferred sequence is an easy manufacture and detect given sequence only, and conventional " screening " method of carrying out known to those skilled in the art.

F. transformant analysis

Generally undertaken by the preliminary evaluation of transformant by the expression that detects selected labelled protein.At GFP is among the embodiment of labelled protein, uses the FITC filter to analyze by fluorescent microscope.After the preliminary evaluation transformant colony, described colony goes down to posterity by picking and expands.The single colony that goes down to posterity from gained by pcr analysis.This technology successfully has been used for mouse and has been detected homologous recombination (Smithies, 1991).The primer that each experiment is used depends on the transgenosis of introducing.

G. homologous recombination

The genomic improvement work of selected domestic animal species has become history and has poured into the focus of research over 15 years.At present, unique available techniques of generation breeding transgenic livestock is pronucleus injection or virus vector.Produce transgenic pig by the pronucleus injection and proved difficulty and invalid method (Wall, 1996), this part be because the character of pronucleus injection technique itself, it causes the unpredictalbe random integration of introducing of DNA and obtains bad result (Pursel etc. in some cases, 1989), and part owing to produce expensive that transgenic pig follows.Though viral transformation ratio pronucleus injection is more effective, this technology still is accompanied by at random and inserts, the technical problems of inlaying and producing replication defect type recombinant viral vector aspect that form of integrating more.Therefore, aspect domestic animal, about the shortage of the complete data of genetically modified suitable expression and adjusting with produce the technological deficiency that transgenic animal followed and combine.

Some shortcoming of pronucleus injection and virus vector can overcome (Koller and Smithies, 1992) by utilizing known homologous recombination technique.This technology is allowed accurate modification odc gene, has solved the problem of position effect and insertion inactivation, and has allowed the deactivation specific genes, and replaced another with a gene.Regrettably, the efficient of this method is too low so that can not be directly used in the embryo, and must use a kind of carrier cell system.This clone of Shi Yonging is that embryonic stem cell (ES) produces the transgenic animal of carrying these changes by the injection of ES blastocyst then because of its easy handling and in external selection so far.Till the present invention, homologous recombination only can be carried out in mouse, allows the domestic animal ES cell that carries out same genetic manipulation because still can not separate.This composition and method make and can cultivate and genetic manipulation domestic animal clone with the quality of ES sample.

Therefore the preferred method of the transmission of transgenosis construct comprises homologous recombination method or " the rejecting technology " used.Be similar to the antisense method, homologous recombination technique depends on the tendency of nucleic acid and complementary sequence base pairing.In this case, base pairing assists two isolated nucleic acid molecule to interact so that chain break and reparation can take place.In other words, to be that sequence homology is attracted to two complementary sequences closely adjacent for the content of " homology " in the described method, and the content of " reorganization " provides a complementary sequence to replace another and formed other sequence owing to the fracture of some key.

With regard to practice, homologous recombination is used as follows.At first, target gene is chosen in host cell.Be comprised in a kind of genetic constructs with described target gene homologous sequence then, and certain sudden change will make described target gene inactivation (terminator codon is interrupted etc.).The homologous sequence of described inactivation sudden change flank is called as " side is positioned at " described sudden change.In the present context, flank only refers to that the target homologous sequence is positioned at the upstream (5 ') and the downstream (3 ') of described sudden change.These sequences should be corresponding with some sequence in the upstream preface of described target gene and downstream.Then described construction is introduced described cell, described cell sequence and described construction are recombinated.

In fact, be not limited to remove to interrupt described gene under the described genetic constructs normal circumstances as carrier.For example, it importantly can be selected recon and therefore comprise the selected marker usually in described construction.This gene is allowed by giving the resistibility to various biological functions and Biocidal medicine, selecting and is integrated described construction and enter cell in its genomic dna.What in addition, expressed exogenous gene also can be favourable in this cell is included in the described construction.Can be by following arrangement:

... carrier * 5 '-flanking sequence * heterologous gene * screenable marker gene * flanking sequence 3 ' * carrier ...

Therefore, use this construction, in substance group incident, may native gene of (i) " rejecting ", but (ii) provide a selection markers to identify this incident and (iii) to introduce transgenosis for expression.

Another kind of improved homologous recombination method comprises use " feminine gender " but selection markers.This mark (but being different from described selection markers) causes the death of the cell of expressing described mark.Therefore, it is used to identify unwanted recombination event.But when seeking to use a kind of selection markers screening homologous recombination, be difficult in initial screening step from from random, the single-minded incident of non-sequence identifies suitable homologous recombination.But these recons also can contain described screenable marker gene and expressing heterologous target protein matter, but do not have needed " rejecting " phenotype most likely.But, but just can mix the recon of described feminine gender selection markers at many screenings of recombination event at random by the selection markers in described construction (but being positioned at outside the described flanking sequence) that connects a feminine gender.But homologous recombination should not introduced the feminine gender selection markers, because it is in the outside of flanking sequence.Use the method example of negative sieve method enrichment homologous recombination to comprise and be directed to gene (Mortensen, 1993 in blocking-up embryonic stem cell or the transformation cell lines; Willnow and Herz, 1994) and produce recombinant virus (Imler etc., 1995) as adenovirus.

Because obviously influencing of the source of the DNA that the frequency of gene targeting is used to lead, similar (isogenic) DNA is helpful with being directed to cell as far as possible in acquisition.An approach finishing this work is by separating the purpose zone from the genomic dna from single colony with long scope PCR.Use long scope PCR, might isolate the fragment of 7-12kb from a small amount of initial DNA.For finishing this work, present inventor's original PGC deutero-cell colony conditions needed of having determined successfully to go down to posterity.As seeing among the embodiment 3, after going down to posterity, might obtain the colony number that 10-20 doubly increases at every turn.Inoculate this work of finishing by tryptic digestion with high-density at fresh feeder layer middle plateform.Yet the purpose zone is very conservative so that can use non-isogenic DNA in some cases.The apoE locus of this situation such as mouse, the present inventor can lead with non-isogenic dna high frequency.

It is the useful technology that a kind of the present invention of being suitable for uses that gene is caught.This technology relates to utilizes the endogenous regulatory region that is present in the described chromosomal DNA to activate alien gene.In this way, when described transgenosis was inserted into a random site, this genetically modified expression was lost or is minimized.Yet, be in external transgenosis and during the endogenous regulatory region of column position, just cause genetically modified expression when homologous recombination occurs in.

H. transgenosis excision

The member of intergrase family is the protein that is attached to the DNA recognition sequence, and discerns with DNA, joint conference, and fracture, the interchain exchange, relevant with reclosing.At present, intergrase family comprises 28 protein, from bacterium, and phage and yeast, they have common constant Histidine-arginine-tyrosine triplet (Abremski and Hoess, 1992).Four systems during eukaryote is used in the most popular site-specific nature recombination system comprises: the Cry-loxP of phage P1 (Austin etc., 1981); The FLP-FRT of cereuisiae fermentum 2 μ plasmids (Audrews etc., 1985); The R-RS of Zygosaccharomyces rouxii (Maeser and Kahmann, 1991) and μ phage gin-gix (Onouchi etc., 1995).Cre-loxP and FlP-FRT system are than the degree height of latter two systems exploitation.The R-RS system is similar to Cre-LoxP FLP-FRT system, only needs described protein and recognition site thereof.Gin recombinase selectivity is regulated the DNA inversion between the recombination site (gix) of two reverses direction and is needed the assistance of three additional factors: negative supercoiling, enhancer sequence and conjugated protein Fis thereof.

The present invention expects to use Cre/Lox site specific recombination system (Sauer, 1993, Gibco/BRL company, Gaithersburg, Md. provides) to rescue concrete gene from genome, and excises concrete transgenosis from described genome.Cre (causing reorganization)-loxP (locus of intersection (x)) recombination system separates from phage P1, and it only needs Cre enzyme and the loxP recognition site on two mating partner molecules (Sternberg and Hamilton, 1981) thereof.The LoxP site is made up of the symmetric 13bp protein bound district of two interval 8bp, and this site is discerned by Cre recombinase (a 35kDa protein).The nucleotide sequence of LoxP (Hoess etc., 1982) and Cre (Sternberg etc. 1986) is known.If two loxP sites are mutual cis, the excision reaction then takes place; And if two sites are trans mutually, then integration incident takes place.Cre albumen catalytic site specificity recombination event.This incident is two-way, i.e. Cre catalysis sequence is inserted in the LosP position or the sequence of excision between two LoxP positions.Therefore, if the construction that inserts also contains the flanking sequence of LoxP position, then with Cre protein, or the process that the proteinic polynucleotide of coding Cre are introduced described cell removes described structural DNA with catalysis.This technology is according to U.S. Patent number 4,959, and 317 implement, and quotes in full to be reference herein.

Studies show that in the initial body that in bacterium Cre excises loxP flanking DNA (Abremski etc., 1983) outside the karyomit(e) in the cell of expressing described recombinase.A subject matter of relevant this system whether in the eukaryotic cell site-specific reorganization can obtain promoting by a kind of bacterioprotein.Yet, Sauer (1987) show described system in cereuisiae fermentum with level of efficiency excision DNA identical in bacterium.

Use the Cre-loxP system, particularly the further research carried out of mouse ES cells system has proved that it is useful that the excision reaction pair produces special transgenic animal.After the homologous recombination, use the disappearance of the loxP flank neo-tk box of Cre mediation to suddenly change to the ES cell with introducing.4 samsaras are to change the allelotrope of rep-3 and mMsh2 locus repeatedly altogether in same cell system for this strategy, and they are and correct the relevant gene (Abuin and Braadley, 1996) of dna mismatch.Equally, by 35S promoter/luciferase gene/loxP/35S promotor/hpt gene/loxP (luc ⁺Hyg ⁺) transgenosis formed is introduced in tobacco.Handle with Cre subsequently and cause hyg gene (luc ⁺Hyg ^s) 50% disappearance (Dale and 0w, 1991).There is the transgenic mice of Ig light chain κ constant region of the neo gene targeting of loxP side position to be bred into the mouse of product Cre to remove described selected marker (Lakso etc., 1996) from its body early embryo.The method that this generality removes selected marker is formed at care with the adjusting tissue of the problem of a colony aspect of new gene introducing and the arguement that the user is proposed.

The desired system that is similar to that uses among the present invention is the FlP/FRT system.This system histone 4 genes in the mouse ES cells of FRT side position neo box that are used to lead, the reorganization by the FLP mediation makes described mark disappearance then.Flp protein can be from the inducible promoter of driving FLP or by using described protein itself to obtain (Wigley etc., 1994).

The present invention also expects to use recombination activation gene (RAG) 1 and 2 to excise concrete transgenosis construct from genome and to save concrete gene from described genome.RAG-1 (GenBank number of registration M29475) and RAG-2 (GenBank number of registration M64796 and M33828) discern single-minded recombination signal sequence (RSSs) and the assembling of catalysis immunoglobulin (Ig) and the needed V of TXi Baoshouti gene (D) J reorganization (Schatz etc., 1989; Oettinger etc., 1990; Cumo and Oetinger, 1994).The proteinic transgene expression of RAG-1 in the non-lymphocyte and RAG-2 is supported V (D) the J reorganization (Oettinger etc., 1990) of intelligencer's substrate.With regard to using among the present invention, purpose transform construction by through engineering approaches to comprise flank RSSs.After the conversion, RAG-1 that the conversion construction of described RSSs inside can be by in transformant and RAG-2 transient expression and from described genomic deletion.

The immortalization of I.PGCs

The description of a relevant immortalization urogenital ridge derived cell aspect of having ready conditions is only arranged so far.Clone behavior in the described cultivation is slightly different with the behavior of non-immortalized cell line, thereby prompting might make described clone have ready conditions immortalization so that it is cultivated, and keeps and genetic manipulation, keeps the ability to function that it forms mosaic simultaneously.The notion of the necrocytosis by having the genitaloid immortalization interference programization that transforms construction results from following observation: Bcl-2 and suppresses vitamin A acid inductive apoptosis (Okazawa etc., 1996) between the differentiation phase at embryonic stem cell; Apoptosis with cultivate in genitaloid death relevant (Pesce and Felici, 1994); The survival (Pesce etc., 1993) that promotes sexual cell by checking programmed cell death with STEM CELL FACTOR and leukaemia inhibitory factor.Therefore, transform the ability that interference program necrocytosis approach should improve the PGCs in maintenance and the genetic manipulation cultivation by having ready conditions.

Transforming gene that exemplifies and construction are listed in following.These genes belong to different functional categories, as the undesired signal transduction, influence the cell cycle, change nucleus and transcribe, and change telomere structure or function, suppress apoptosis, or produce the multiple-effect effect.Will be understood that listed gene only is the oncogene that can use in the present invention, the type of the tumour repressor of mutagenesis and other transforming gene construction and the factor for example.Other transforming gene and construction should be that this area routine techniques personnel know.

Numerous protein has demonstrated the inhibition apoptosis, i.e. the necrocytosis of sequencing.This type of proteinic representative is bcl-2 and comprises Bcl-x1, Mcl-1, Bak, A1, the family member of A20, and interleukin-1 ' beta '-conversion enzyme and family member.Promoted genitaloid survival (Pesce etc., 1993) owing to demonstrated the apoptotic somatomedin of inhibition already, this proteinoid is particularly preferably in using among the present invention.The preferred protein that uses is that bcl-2 (is different from bcl-1, cyclin D1; Genbank number of registration: M14745, X06487).The overexpression of this oncogene is at first found in t cell lymphoma.It brings into play function as oncogene by combination and deactivation bax (a kind of protein in the apoptotic pathways).

Except suppressing apoptotic protein, it is reported that numerous protein can not promote apoptosis.Comprising p53, retinoblastoma gene (Rb), Wilm tumour (WT1), bax α, interleukin 1 b-conversion enzyme and family thereof, MEN-1 gene (karyomit(e) 11q13), neurofibroma, Class1 (NF1), cdk inhibition p16, colorectal carcinoma gene (DCC), familial causes adenoma gene (FAP), polyoma repressor gene (MTS-1), BRCA1, BRCA2.

Preferably p53 and retinoblastoma gene.The cancer report of most forms has the p53 sudden change.The inactivation of p53 causes promoting apoptosis.Along with the forfeiture of this function, cancer cells development and can final cell death in tumour generates.The tabulation of a weak point of cancer of finding in p53 and sudden change is: and ovary (Genbank number of registration: S53545, S62213, S62216); Liver (Genbank number of registration: S62711, S62713, S62714, S67715, S72716); Stomach (Genbank number of registration: S63157); Colon (Genbank number of registration: 63610); Bladder (Genbank number of registration: S85568, S85570, S85691); Lung (Genbank number of registration: S41969, S41977); Neurospongioma (Genbank number of registration: S86807, S85712, S85713).

There is the tumour repressor of many known oncogenes and sudden change to work by the perturbation signal transduction.This class line-up of delegates is Tyrosylprotein kinase (tenuigenin and cytolemma correlation form include), as Scr family, and Jak/Stats, Ros, Neu, Fms, Ret, Abl and Met.Other member in this type of is a serine/threonine kinase, as Mos, and Raf, protein kinase C (PKC) and PIM-1.Another family that belongs to this type of is somatomedin and acceptor, as Thr6 PDGF BB (PDGF), rhIGF-1 (IGF-1), IRS (IRS-1 and IRS-2), Erb family, Urogastron (EGF), tethelin, pHGF (HGF), Prostatropin (bFGF), and corresponding growth factor receptors.Little GTP enzyme or G albumen also belong to this type of, and with ras family, rab family and Gs-α are representative.Receptor type tyrosine phosphatase IA-2 also is this type of proteinic member.

The preferred in the present invention member's example that uses is Neu, also is called Her2, also is called erbB-2 (Genbank number of registration: M11730, X03363, U02326 and S57296).The oncogene of finding in mammary cancer also is found in other cancer form.A member in this seemingly described receptor tyrosine kinase family.Preferred pHGF (HGFr in addition; Genbank number of registration: U11813), also be called the dispersion factor acceptor.This may be the example (or endogenous existence or express from recombinant adenovirus) in the acceptor, and it is used to stimulate target cell group's propagation.Other preferred member be type-1 insulin like growth factor acceptor (Genbank number of registration: X04434 and M24599) and GTP enzyme Gs α (Genbank number of registration: X56009, X04409).Gs α is relevant with the pituitary tumor of secretion tethelin, but irrelevant with other neuroendocrine or endocrine tumors.

Existing transforming gene influences the description of cell cycle.The protein that belongs to this type of is cyclin-dependent protein kinase (cdk), the A-E class; Member such as cyclin D with cyclin family.The example of Shi Yonging is a cyclin D1 in the present invention, also is called PRAD, also is called bcl-1 (Genbank number of registration: M64349 and M73554).As a kind of oncogene, it is main relevant with parathyroidoma.

Many transforming genes have been described to transcribe and bring into play its influence by changing nucleus.This genoid comprises the Myc family member, comprising c-myc, and N-myc, and L-myc; The Rel family member comprises NF-κ B; CMyb, Ap-1, fos and jun, insulinoma Related cDNAs (IA-1), Erbb-1 and PAX gene family.The example that uses among the present invention is c-myc (Genbank number of registration: J00120, K01980, M23541, V00501, X00364.

A protein that has been included in recently in the cell transformation is Telomerase.The assembling of Telomerase and telomere and keep relevantly, telomere is positioned at chromosomal end.At present do not know still Telomerase is how to bring into play function in conversion.

Some transforming genes have the multiple-effect effect.Some protein wherein are virus protein such as SV40 and polyoma large T antigen, SV40 responsive to temperature type large T antigen, adenovirus E 1 A and E1B protein and papillomavirus E6 and E7 protein.SV40 large T antigen (TAG preferably in this type of; Genbank number of registration: J02400).Also has preferably responsive to temperature type large T antigen.

The generation of the non-rodent of III transgenosis

As mentioned above, the condition of generation transgenic rodent is not suitable for and produces other genetically modified organism.Method of the present invention at first makes it possible to from each species quick, reliably and at low cost produces non-rodents transgenic animal.

A. animal species

The present invention can be used to produce transgenic animal from any non-rodent kind.It is preferred among the present invention that what use is Mammals, and more preferably pig, ox, sheep and billy goat.

B. consideration convey moves

The existing method that the domestic animal center shifts is next by deriving of the method for McGrath and Soler (1983) exploitation.Donor embryo and unfertilized acceptor ovocyte are handled with the cytoskeleton inhibition, and a micro pipette is inserted in the described ovocyte, and mitosis metaphase, karyomit(e) was moved out of together with the membrane-bound tenuigenin of a part.Successful stoning monitoring method comprises that direct viewing is chromosomal and shifts out (Stice and Robl 1998), uses DNA specificity fluorescent dyestuff bisbenzimide substantive dyeing (Tsunoda etc., 1988; Prather and First1990a; Westhusin etc., 1990) or fixing described by the part of enucleation oocyte and suppose identical stoning efficient is arranged all the other ovum (Willadsen 1986; Prather etc., 1987; Smith and Wilmut 1989).Then, architomy ball of donor embryo (or its part) is sucked micro pipette and pushes perivitelline space, adjacent with described non-nucleus egg mother cell.Next step is at two cells of perivitelline space scope endomixis.This process can be finished with Sendai virus (Graham1969) or electricity consumption fusion (Berg1982) in some kind.

The efficient of described pitting method can reach 100% (Tsunoda etc., 1988 when described karyomit(e) is directly or indirectly observed; Stice and Robl 1988; Prather and First 1990a), and when the karyomit(e) that shifts out only was the basis with the position of first polar body, the percentage ratio of enucleation oocyte is lower, and (Willadsen 1986; Prather etc., 1987,1989a).

Activation is considered to merge generation simultaneously with electricity.Know that already electricimpulse is a kind of effective monogenesis reagent (Whittingham 1980) in mouse.Electricity activated special mechanism it be unclear that, but it can with the depolarize of film after electric inductive hole forms and the seepage relevant (Whittingham 1980) of calcium.With regard to fusion, electric inductive activates has very big difference with different research.The factor that influences activity ratio has many, comprises the ovum age and the species of ovocyte, the type of incubator and use the medium of described pulse, and the type of pulse (Collas etc., 1989; Ozil 1990).

With as pig, ox, the built-in problem that sheep and goat is studied is to detect and obtain kind of a system to transmit a needed time span when using normal diploid host embryo to produce mosaic.Whether can increase offspring's effect for measuring described ES or EG, can use two kinds of methods: consideration convey moves and uses tetraploid host embryo to produce mosaic.In the situation that consideration convey moves, detect the totipotency of described cell, and during with the tetraploid embryo, detect the versatility of described cell.

The basic skills that consideration convey moves comprises that acquisition is unicellular and merges them in the acceptor ovum of stoning.The nucleus of this effective transfer donator cell enters in the described recipient cell kytoplasm, if can success, then sequencing and instruct new embryo's growth subsequently once more be equal to that embryo who obtains described cell in described embryo's heredity.The most extreme example of these technology potentiality by report such as Wilmut (1997), shows that nucleus and the Adult Mammals epithelial cell from embryo fibroblast can instruct normal development in sheep.Though this consideration convey technology of moving can not show a candle to the advanced person in the pig, the birth report (Prather etc., 1989) of the success of the nucleus of existing use 4 somatic embryos.

Donor (Cherny and Merei, 1994 of nuclear transplantation successfully have been used as from the PGCs of tire tissue collecting; Delhaise etc., 1995; Lavoir etc., 1997; Strelchenko, 1986).In pig, proved already that the PGCs of previous cryopreservation can be by the nuclear donor that is used as of success, and made the nucleus reprogramming and the spilting of an egg to 4 cell stages (Liu etc., 1995).In addition, Ouhibi etc. (1996) report consideration convey moves the derive reprogramming of pig cell center of the ICM that cultivates the back.Regrettably, described embryo participates in the ability not research as yet of normal development.

In the recent research in ox, Laboir etc. (1997) report is when the ovogonium of collecting from female embryo (50-70 days pregnant ages) is used as nuclear donor, and 9-13% grew to blastocyst stage in the splitted nuclear transfer embryo.Though the calf that does not have output to live, one has only four embryos' animal that a unusual gestation has taken place.This gestation is induced labor during in the 43rd day after detecting less than heartbeat, and genetic analysis shows that this embryo and described donor ovogonium are hereditary consistent.Use similar results from the hero of ox and female embryo's PGCs by reports (Moens etc., 1996) such as Moens.Strelchenko (1986) observes instructs to grow from endorsing of the ox PGCs that cultivates did not have the unusual phenomenon prompting of being reported of obvious embryo by 60 days, when PGCs is in the cultivation, compare with new isolating PGCs, the nucleus of potentiality that can increase the nuclear of described cell changes.

Increasing described ES clone is to carry out the ES injection cell with the tetraploid embryo as the host to the another kind of method of mosaic embryo's effect.Make in this way, developmental tetraploid cell is limited in placenta tissue, even and diploid ES cell does not form all, also form suitable embryo's big portion.When employed mouse primary cell line produces the time limit offspring (Nagy etc., 1990) who promptly dies young soon after the birth, use other ES clone to cause having the survival of 100% ES effect to the mosaic (Ueda etc., 1995) of growing up and normally breeding.In pig, when flocking together with the diploid blastomere, the ability that the tetraploid embryo forms the mosaic blastocyst obtains proof (Prather etc., 1996).

C. blastocyst injection

In this technology, the blastocyst stage embryo shifts out from the female body of becoming pregnant.PGC derived cell colony is dissociated into unicellular, and cultivates with 2-5 blastocyst stage embryo.Inject the segmentation cavity that described mixture enters embryo in the growth then.After the injection, described embryo is placed in the incubator and makes its recovery.Then, than donor embryo late (postponement) 24 hours, the embryo of recovery returned be in estrous acceptor.An example is to use the 6th day donor embryo and the 5th day acceptor.After the transfer, monitor described animal every day.By turning back to oestrus and ultrasonic definite conceived situation.

D. assemble with body early embryo

Making chimeric another approach is to make the PBC derived cell and assemble than body early embryo (the particularly embryo before the 8 cell compactnesses).The method of finishing this process is that 10-12 PGC derived cell of injection enters 8 cell stage embryos' perivitelline space, and cultivate blastocyst stage and mix among the described ICM, or shift out 8 cell stage embryos' zona pellucida and place described embryonic cell with 8-12 PGC derived cell close apposition with definite described PGC cell.Make described fetal development in the acceptor of blastocyst stage with suitable period of determining described PGCs and mix ICM and being transferred to the described oestrus cycle.

E. tetraploid embryo

Enhancing PGC derived cell is to use the host of tetraploid embryo as the injection of PGC derived cell to another preferred method of described mosaic embryo's effect.Use this method, make developmental tetraploid cell be limited in described placenta tissue, and described diploid PGC cell forms the major portion of (if not all) suitable placentas.(1996) such as tetraploid embryo's production method such as Prather are described.Basically, two somatic embryos are to collect from the operation on fallopian tube after oestrus detection and mating.The embryo is by equilibrium and fusion.After the fusion, the embryo is placed in the Whitten substratum and cultivated 6 days at 39 ℃.In this period, the tetraploid embryo is used as the host embryo.Behind 10-15 PGC derived cell of injection, the embryo will be transferred to the synchronization acceptor and make it to grow amortization period.

F. transgenic animal are analyzed

In a preferred embodiment of the invention, collect the embryo from the animal of becoming pregnant in early days, and detect described genetically modified expression.In particularly preferred embodiments, the GFP labelled protein is included in and has at least in a kind of selected genetically modified construction that adds.In dissecting microscope, carry out under the luminescent lamp dissecting described embryo to shift out genital ridge after Preliminary detection determines GFP expression degree.Detect sexual gland (reproduction) ridge once more with fluorescent microscope.GFP is accredited as the male primordial germ cells and is identified, and shows described by the be formed with effect of transformation cell lines to germline mosaic.The described genital ridge that dissociates, and separate GFP and under fluorescent microscope, check, and the ratio of calculation expression GFP cell.After fluorometric analysis, preserve whole residue tissues for the PCR genetic analysis of DNA extraction and described DNA and genome Southern analysis in order to confirm described genetically modified existence.

In case identified the embryo who contains transgenosis GFCs, made all the other pregnant animal gestation to stopping.At amortization period, collect placenta tissue and get the umbilical cord sample for genome analysis from every birth porkling.After two to three days, collect little tissue sample with DNA isolation with carry out Southern Analysis and Identification transgenic animal.Described animal is continued to grow up to body surface by hair, get blood this moment and be used for DNA and separate and identify transgenic animal.Keep the usefulness of whole certified transgenic animal for further research.

Still the animal among observing will be held reproduction age to determine the effect degree of described sexual cell to the PGC derived cell.The effect of described sexual cell will be by breeding and determining by collecting seminal fluid sample (if transgenic animal are male).Handle described sample separating pure sperm group, and DNA isolation is carried out PCR and Southern and analyzed determining whether described transgenosis can detect in described sperm, thereby show kind of potentiality that are transmission.

By the genetically modified expression of the described GFP of fluoroscopic examination, and confirm it by the Northern method.In all cases, at first detect tissue with described genetically modified existence certainly by Southern, then by Northern determining described gene transcription, and determine described proteinic translation by fluorescent method then.Under the situation beyond the GFP, follow same order, different is that final step carries out the PAGE protein analysis, carries out Westerns subsequently, or carries out biological detection.

The heritability of described gene is confirmed by germline mosaic and test animal mating, analyzes to going out cub's sampling by the Southern to tissue and blood DNA.In this stage, transgenic animal should right and wrong chimeric and be hemizygote, therefore if heredity just might detect described transgenosis.For confirming that described transgenosis can pass to the next generation, confirmed to express in a suitable manner described genetically modified hemizygote transgenosis and further bred, and analyzed its transgenosis heredity of young baby that produces.

IV. growth factor gene

Further content relation of the present invention is to separated DNA section and recombinant vectors, its bovine leucosis supressor (LIF) of encoding, pig ciliary neurotrophic factor (CNTF), with pig lipophorin-E (Apo-E), and by using the dna technique establishment and using and express ox LIF, the recombinant host cell of pig CNTF and pig Apo-E.

Additional aspects of the present invention relate to separated coding pig leukaemia inhibitory factor (LIF; SEQID NO:7) and pig ciliary neurotrophic factor (CNTF; SEQ ID NO:8) DNA section, they have been optimised to reach high expression level in yeast.This is to select with the codon in the performance yeast by changing dna sequence dna, and the aminoacid sequence that does not change the protein of generation is finished.When the protein that produces from these sequences enters recombinant host cell in combination, can give primordial germ cells suitable growth-promoting activity, separable pig LIF or pig CNTF from these cells.

The present invention relates to from ox (LIF) and pig (CNTF and Apo-E) separated DNA section, they do not contain genomic dna and can give primordial germ cells suitable growth-promoting activity when combination enters primordial germ cells or recombinant host cell, ox LIF, pig CNTF and pig Apo-E can be separated from described primordial germ cells or reconstitution cell, and join then in the suitable growth medium.Term used herein " growth-promoting activity " refers to make primordial germ cells to grow in the mode that keeps its cell undifferentiated state, and gives them and be easy to transform the ability of freezing and long-term cultivation.

As used herein, term " DNA section " refers to the isolating dna molecular that does not contain the total genomic dna of concrete species.Therefore, it is separated or be purified and do not contain total genome ox DNA that the DNA section of coding ox LIF also refers to contain the sequence of coding ox LIF, and the DNA section of coding pig CNTF or Apo-E refers to contain the DNA section of pig CNTF or Apo-E encoding sequence, but separated or be purified and do not contain total genome pig DNA.Be included in this term " DNA section " be DNA section and this class section than small segment, and comprise recombinant vectors, for example comprise plasmid, clay, phage, virus etc.

Equally, the ox LIF that contains isolating or purifying, a DNA section of pig CNTF or pig Apo-E gene is meant and comprises ox LIF, pig CNTF or pig Apo-E gene coded sequence and (in some aspects) are regulated the DNA section of sequence, and described DNA section is separated and break away from other gene that exists naturally or protein coding sequence basically.In some aspects, term " gene " simply is used in reference to a kind of functional protein, the coding unit of polypeptide or peptide.As should be by understood by one of ordinary skill in the art, this functional term have both comprised genome sequence, and the cDNA sequence comprises that again expression maybe can be adapted to marking protein, polypeptide or peptide than the unskilled labourerization constant gene segment C.

" break away from other encoding sequence basically " and mean goal gene that (in this situation is ox LIF, pig CNTF or pig Apo-E gene) form the meaningful part of described DNA section coding region, and mean the coding DNA that described DNA section does not contain the nature existence, as macrochromosome fragment or other functional gene or cDNA coding region.Certainly, this is meant that described DNA section is original isolating, and is added to gene or coding region in the described section after not arranging manually.

In specific embodiments, the present invention relates to separated DNA section and mixed coding ox LIF, the recombinant vectors of the dna sequence dna of pig CNTF or pig Apo-E gene, described gene has comprised in its aminoacid sequence and ox (LIF) or pig (CNTF, Apo-E) Dui Ying SEQ IDNO 2 (ox LIF), the continuous amino acid sequence of SEQ ID NO 4 (pig CNTF) and SEQ ID NO 6 (pig Apo-E).

Nature, at DNA section or vector encoded total length ox LIF, pig CNTF or pig Apo-E protein or plan are used for expressing described ox LIF, when pig CNTF or pig Apo-E protein, most preferred sequence is basically as at total length continuous sequence SEQ ID NO 2 (ox LIF), those illustrated sequences among SEQ ID NO4 (pig CNTF) and the SEQ ID NO 6 (pig Apo-E), and be coding has kept the protein (for example can measure cultivating genitaloid energy) to genitaloid growth-promoting activity in cultivation those sequences.

Sequence of the present invention will correspond essentially to SEQ ID NO 2 (ox LIF), SEQ ID NO 4 (pig CNTF) and SEQ ID NO 6 (pig Apo-E), sequential portion, and contain relatively small amount with SEQ ID NO 2 (ox LIF), the amino acid of the inconsistent or biological function non-equivalence of SEQ ID NO 4 (pig CNTF) and SEQ IDNO 6 (pig Apo-E).Term " the biological function equivalence " be this area fine understanding and advance one to be defined (seeing the X joint) in this article.

Therefore, contain between about 70% and about 80%; Or more preferably from about between 81% and about 90%; Even more preferably from about amino acid between 91% and about 99% and SEQ ID NO 2 (ox LIF), SEQ IDNO 4 (pig CNTF) and SEQ ID NO 6 (pig Apo-E) amino acid sequence consistent or function equivalence should be basically at SEQ ID NO 2 (ox LIF), the sequence of describing among SEQ ID NO 4 (pig CNTF) and the SEQ ID NO 6 (pig Apo-E).

In some other embodiment, the present invention relates to separated DNA section and recombinant vectors, being included in has from SEQ ID NO 1 (ox LIF) SEQ ID NO 3 (pig CNTF) and SEQ ID NO 5 (pig Apo-E), " optimization " pig leukaemia inhibitory factor (LIF in its sequence; SEQ ID NO 7) and the continuous kernel acid sequence of " optimization " pig ciliary neurotrophic factor (CNTF SEQID NO 8).This definition is with same meaning use as described above, and mean and correspond essentially to SEQ ID NO 1 (ox LIF), SEQ ID NO 3 (pig CNTF) and SEQ ID NO 5 (pig Apo-E), the continuous kernel acid sequence of SEQ ID NO 7 (" optimization " pig leukaemia inhibitory factor) and SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor) sequential portion also contains relative few and SEQ ID NO 1 (ox LIF), SEQ ID NO3 (pig CNTF) and SEQ ID NO 5 (pig Apo-E), the codon of non-equivalence on the inconsistent or function of SEQ ID NO 7 (" optimization " pig leukaemia inhibitory factor) and SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor) codon.The DNA section that coding presents the protein of primordial germ cells growth-promoting activity should be most preferred.Term used herein " codon of equivalence on the function " is meant the same amino acid whose codon of coding, as six codons of coding arginine or Serine, and refers to upward amino acid code of equivalence of encoding human.Table 5 and table 6 during ginseng sees next section.

Ox LIF (SEQ ID NO 49), the genome sequence of pig CNTF (SEQ ID NO 50) and pig Apo-E (SEQ ID NO 51) are also open at this paper.The exon of ox LIF is corresponding to Nucleotide 1214-1298,3010-3188 and 3949-4359 among the SEQ ID NO 49.The exon of pig CNTF is corresponding to Nucleotide 392-505 and 1768-2256 among the SEQ ID NO 50.The exon of pig Apo-E is corresponding to Nucleotide 832-858,1663-1728,2473-2662 and the 3037-3879 of SEQ ID NO 51.Described exon is corresponding to the coding region, and in some cases corresponding to 5 with (or) 3 non-translational regions.

What will also be understood that is that amino acid and nucleotide sequence can comprise other residue, as other N-or C-end amino acid or 5 or 3 sequences, and be described as one of sequence disclosed herein basically, as long as described sequence satisfies above-mentioned standard, comprise and protect the protein biology activity that relates to protein expression.Additional end sequence is applied to especially for example can to comprise that the various non-coding flanking sequence of coding region 5 or 3 maybe can comprise the nucleotide sequence of various slotting sequences (being the known intron that is present in gene inside).

Intron or the flanking region outer merger of genetic code (and allow) contain between about 70% and about 79%; Or more preferably from about between 80% and about 89%; Even more preferably from about Nucleotide between 90% and about 99% and SEQ ID NO 1 (ox LIF), SEQ ID NO 3 (pig CNTF) and SEQID NO 5 (pig Apo-E), the identical sequence of Nucleotide of SEQ ID NO 7 (" optimization " pig leukaemia inhibitory factor) and SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor) should be basically at SEQ ID NO 1 (ox LIF), the sequence of describing among SEQ ID NO 3 (pig CNTF) and the SEQ ID NO 5 (pig Apo-E), SEQ ID NO 7 (" optimization " pig leukaemia inhibitory factor) and SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor).Substantially the same in SEQ ID NO 1 (ox LIF), SEQ ID NO 3 (pig CNTF) and SEQ ID NO 5 (pig Apo-E), the sequence of describing among SEQ ID NO 7 (" optimization " pig leukaemia inhibitory factor) and the SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor) also can be defined as on the function under relative tight condition can with contain SEQID NO 1 (ox LIF), SEQ ID NO 3 (pig CNTF) and SEQ ID NO 5 (pig Apo-E), the sequence of the nucleic acid segment of SEQ ID NO 7 (" optimization " pig leukaemia inhibitory factor) and SEQ IDNO 8 (" optimization " pig ciliary neurotrophic factor) complementary sequence hybridization.Suitable relatively compact hybridization conditions should be that those skilled in the art are well-known.

Nature, the present invention also comprises with SEQ ID NO 1 (ox LIF), complementary or the basic complementary DNA section of the sequence of describing among SEQ ID NO 3 (pig CNTF) and the SEQ ID NO 5 (pig Apo-E), SEQ ID NO 7 (" optimization " pig leukaemia inhibitory factor) and SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor).The nucleotide sequence of " complementation " is can be according to standard Watson-Crick principle of complementarity paired sequence.Term " complementary sequence " means complementary nucleotide sequence basically as used herein, as can be estimated according to above-described equal nucleic acid corresponding relation, or as according to can be under tight relatively condition and SEQ ID NO 1 (ox LIF), SEQ ID NO 3 (pig CNTF) and SEQ ID NO 5 (pig Apo-E), the nucleic acid segment hybridization of SEQID NO 7 (" optimization " pig leukaemia inhibitory factor) and SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor) is defined.

Nucleic acid segment of the present invention, no matter the length of its encoding sequence own as, can with other dna sequence dna such as promotor, polyadenylation signal, additional restriction enzyme sites, multiple clone site, knots such as other coding region, their entire length can alter a great deal thus.Therefore, expectation can be used the nucleic acid fragment of almost appointing length, and preferred convenient operation of total length and making in desired recombinant DNA side are used for limiting.For example, can prepare and comprise with SEQ ID NO 1 (ox LIF), SEQ ID NO 3 (pig CNTF) and SEQ ID NO 5 (pig Apo-E), the nucleic acid fragment of the continuous section that SEQID NO 7 (" optimization " pig leukaemia inhibitory factor) or complementary consistent with SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor) is short, 14 Nucleotide and can preparing according to appointment up to about 10,000 or about 5,000 base is to length, and about in some cases 3,000th, and is preferred.Total length is about 1,000, and is about 500, about 200, about 100, and about 50 bases also are contemplated to be useful to the DNA section (section that comprises intermediate length) of length.

Should be understandable be that " intermediate length " means institute and quote a length between the scope in context, as 14,15,16,17,18,19,20, etc.; 21,22,23, etc.; 30,31,32, etc.; 50,51,52,53, etc.; 100,101,102,103 etc.; 150,151,152,153, etc.; Comprise and run through 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10, whole integers of 000 scope, up to and comprise sequences such as about 12,001,12,002,13,001,13,002.

Will also be understood that, the invention is not restricted to SEQ ID NO 1 (ox LIF), SEQ ID NO2, SEQ ID NO 3 (pig CNTF), SEQ ID NO 4, SEQ ID NO 5 (pig Apo-E), SEQ ID NO 6, the concrete nucleic acid aminoacid sequence among SEQ ID NO 7 (" optimization " pig leukaemia inhibitory factor) and the SEQ ID NO 8 (" optimization " pig ciliary neurotrophic factor).Therefore, but recombinant vectors and separated DNA section each do not wait comprise ox LIF, pig CNTF, pig Apo-E and pig LIF coding region itself, be loaded with the selected coding region that changes or modify in the basic coding district, or their codifieds comprise ox LIE, pig CNTF, biological function equivalent protein or polypeptide that the big polypeptide of pig Apo-E and pig LIF coding region or codified contain the variant aminoacid sequence.

DNA section of the present invention comprises the ox LIF of biological function equivalence, pig CNTF, pig Apo-E and LIF protein and peptide.This type of sequence can be used as and knownly exists in the nucleotide sequence naturally and therefore occur in codon Feng Yu in the coded protein and the result of function equivalence and producing.Perhaps the protein of function equivalence or peptide can produce by using recombinant DNA technology, and wherein the change of protein structure can be based on the consideration of reformed amino acid properties and by the workerization.Can draw the change that designs by the people by using side-directed mutagenesis, for example draw to proteantigen or to detection ox LIF, pig CNTF, the improvement of pig Apo-E and pig LIF muton is to measure genitaloid growth-promoting activity on molecular level.

If need, also can prepare and melt albumen and peptide, for example, at ox LIF, pig CNTF, pig Apo-E and pig LIF coding region and other have the protein of required function or peptide to be arranged in zone in the same ceneme, as be purifying and immunodetection purpose (for example protein can be respectively by affinity chromatography and enzyme labelling coding region and purifying).

V. the clonal growth factor and growth factor receptor gene

The present invention's expectation is by the clonal growth factor and growth factor receptor gene or cDNA in the non-rodent zooblast, and particularly, carcinostatin M (OSM), glycoprotein 130 (GP130), eight aggressiveness are carried down and are recorded the factor 4 (Oct-4), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), lipophorin-E (Apo-E), ciliary nerves growth factor ' alpha ' acceptor (CNTFr-α) and leukaemia inhibitory factor acceptor (LIFr).The zooblast of desired use includes, but are not limited to ox, pig, sheep and billy goat cell.

The technology of protein production those skilled in the art through using is in order to obtain described proteinic what is called " reorganization " version, to express it and obtain described protein from this type of cell in reconstitution cell at present.These technology based on from dna library " clone " go out this protein DNA molecule of coding, promptly be different from other DNA concrete dna molecular partly based on acquisition.This can be by for example cloning the cDNA molecule, or clone gene group sample dna molecular is finished.

First of this clone side is the suitable dna library of screening, and Tathagata is from the genome or the cDNA storehouse of selected non-rodent zooblast.Described screening can be to be a kind of expression screening side that utilizes at described proteinic antibody or the active detection of utilization.For example antibody screening side is that very rule are used.Perhaps, screening can be based on the hybridization of oligonucleotide probe, the consideration of the described proteinic aminoacid sequence of described probe portion or the coding related protein gene dna sequence dna and design.The operation of this type of screening side is that those skilled in the art are well-known and be described in detail (this paper be cited as with reference to) in the scientific literature of (1989) such as for example Sambrook.And because the present invention comprises clone gene group section and cDNA molecule, just as is known to the person skilled in the art, expectation can be used suitable genomic clone side.

VI changes codon and selects to make the genetic expression maximization

The change of expressing in the present invention in the transgenosis of appointing can be formed at the described transgenic sequence of change to accord with the codon selection of selected non-rodent host species.Pertinent data (Bennetzen and Hall, 1982 known in the art that the codon of various biologies is selected; Ikemura, 1981a, 1981b, 1982; Grantham etc., 1980,1981; Wada etc., 1990; Wherein every piece of reference quotes in full at this and is reference).And unrestricted, table 5 provides relevant ox with table 6 with wieldy form as an example, the capsule information of the preferential codon that uses of pig and sheep.Table 5 provides a codon tabulation, and this tabulation is preferred for " oxization " of the present invention " pigization " and " sheepization " construction.Table 6 only is to comprise U (uridylic) but not the identical data of T (thymus pyrimidine), is the convenient usefulness of reference mutually.

Table 5 ox, the codon that pig and sheep are preferably used

¹-GCA is least preferred L-Ala codon in sheep, and few the use

²-TTG is the 3rd in pig, and CTT is the 4th a leucine codon that most preferably uses

³-Niu is the first preferred AGC on TCC; Sheep is the 3rd preferred TCA on AGT

⁴-sheep is the second preferred ACT on ACA

The codon that is positioned at the left side is the codon that most preferably uses, and then preferred to the right usability reduces.

The few codon that uses of double underline codon representative.

The RNA codon that table 6 ox, pig and sheep are preferably used

¹-GCA is least preferred L-Ala codon in sheep, and few the use

²-UUG is the 3rd in pig, and CUU is the 4th a leucine codon that most preferably uses

³-Niu is the first preferred AGC on UCC; Sheep is the 3rd preferred UCA on AGU

⁴-sheep is the second preferred ACU on ACA

The few codon that uses of double underline codon representative.

Data study in his-and-hers watches 5 and the table 6, those skilled in the art are not difficult to find out described ATA, CTA, TTA, CGT, TCG and GTA (or AUA, CUA, UUA, CGU, UCG or GUA) codon is the ox of the embodiment of the invention, should be changed into preferred codon in pig or the sheep.Instruct as generality, those codons in listing in the 5th and the 6th row represents the experimenter in establishment " oxization ", " pigization ", or the codon that preferably changes during " sheepization " gene; The codon of listing in the 4th row is being created " oxization ", " pigization ", or during " sheepization " gene also through being to answer reformed codon; The codon of listing in the 3rd row can or can not be changed, and depends on desired number that changes altogether and the concrete amino acid that will be encoded.Listing in those codons in the 1st and the 2nd row when appearing at the wild-type transgenic sequence in the time, generally is with being also should changing of fitting, and has only a kind of selection in non-two available codons.Yet,, particularly in two codons, have only a kind of situation of selection with codon yes a kind of useful selection that the codon of the 1st row is replaced the 2nd row.Provide these data, will be understood that now, no matter may locate, the general codon of wishing to draw row 1 drawing when changing to described transgenic sequence.

Aforementioned discussion, expectation to the change of codon about 10% will produce the useful increase of expression level and therefore this genoid sequence belong to the scope of the invention.Codon in the described transgenic sequence is about 15%, 20%, 25%, or 30% change also is considered to useful, and the transgenosis that changes among the present invention comprises that those belong to the gene order in the aforementioned range.

In certain embodiments, drawn the character that codon changes, even to carry out 10% change in described genetically modified codon is selected for use also may be unnecessary.For example, if each in the codon of 10 least preferred uses replaced with the codon that most preferably uses in the gene of selected non-rodent species, then expect to produce sequence and in the cell of selected non-rodent species, can reach suitable expression.Follow some other when changing when making these variations, the change of 7,8 and 9 codons of then expecting to have an appointment at least in these codons will be enough to the expression that causes a transgenosis to be improved.As mentioned above, leucine is preferably by CTG, CTC, CTT or TTG coding; Xie Ansuan is preferably encoded by GTG; And Isoleucine is preferably encoded by ATC.

Though existing about 4-5, about 10, about 20 or the sequence that changes of about 30-35% codon generally be preferred, as if need, one change has no reason not do.Therefore, the gene order that the present invention changed can be about 40%, 50%, 60% in total length password subarea, even contain the sequence that is changed codon on the codon position of 70% 80-90%.

The VII protein purification

Of the present inventionly advance a content and relate to the purifying of recombinant heterologous recombinant protein matter, and in specific embodiments, relate to the basic purifying of recombinant heterologous recombinant protein matter.Term used herein " the recombinant heterologous recombinant protein matter of purifying " means can be from the isolating recombinant heterologous recombinant protein matter of a transformed host group thing, wherein recombinant heterologous recombinant protein matter can be purified to corresponding to its natural acquisition state, promptly in the case, corresponding to (for example breast) in its natural product or a purity of cell extract.Therefore, the recombinant heterologous recombinant protein matter of purifying also refers to the recombinant heterologous recombinant protein matter of dissociating and from its naturally occurring environment.

Generally speaking, " purifying " should refer to be extracted the recombinant heterologous recombinant protein matter group thing of various non-host cell compositions.Spendable various technology should be well-known to those skilled in the art in the protein purification.For example these technology comprise and use ammonium sulfate, PEG, antibody etc. or precipitate centrifugal then by thermally denature; Chromatography is rapid as ion-exchange, gel-filtration, and reversed phase chromatography, hydroxylapatite, phytohemagglutinin and other affinity chromatography are rapid; Isoelectrofocusing; Gel electrophoresis; And the group of these and other technology.

But the side that presents low relative purity reclaims aspect the protein or is protecting some advantage aspect the expressed protein active total.The product of deactivation also is being useful aspect the antibody generation for example in certain embodiments.

The partial purification recombinant heterologous recombinant protein matter component of Shi Yonging can be by above-mentioned one of rapid or group is handled transformed host product (for example breast) or cell extract obtains in these embodiments.With the corresponding rapid replacement of improvement some also to be supposed to suddenly be useful.For example, think that carrying out cation exchange column chromatography with the FPLC device generally will cause purifying than the higher multiple of same technology that uses the low pressure chromatographic system.

VIII biological function Equivalent

As mentioned above, can in the transgenic protein structure, make the molecule of modifying and changing and obtain similar or the feature that more needs.For example, in protein structure, some amino acid can be replaced by other amino acid and not have the forfeiture of perceptible oligosaccharides working ability.Since just protein interactions ability and property definition this proteinic biological function activity, can in a protein sequence (or its basic dna encoding sequence) certainly, carry out some amino acid replacement and obtain the protein of similar (excitement) character.Accordingly, can utilize protein or the polypeptide that same consideration is created counteracting (antagonism) character.Therefore, the present invention is desired is can make various changes and do not have its biological applications or active perceptible forfeiture in transgenic protein or peptide (or basic DNA).

With regard to function equivalent, skilled experimenter will also be understood that the notion that is implied in the definition of biological function equivalent protein or peptide is to make in a qualifying part of described molecule that to change and cause having the change amount of the biologic activity of equal value of acceptable level be limited.Therefore, biological function peptide of equal value is defined as wherein those peptides that some (non-big or all) amino acid can be replaced at this paper.Certainly, there are the different many distinct in nature protein of replacing (peptide) can press manufacturing of the present invention and use easily.

Should be well understood that also this type of residue generally can not change when some residue demonstrates the biological property of protein or peptide or textural property very important the residue of reactive site (for example).

For example, conservative replacement well-known in the art comprises that the change L-Ala is to Serine; Arginine is to Methionin; L-asparagine is to glutamine or Histidine; Aspartic acid is to L-glutamic acid; Halfcystine is to Serine; Glutamine is to l-asparagine; L-glutamic acid is to aspartic acid; Glycine is to proline(Pro); Histidine is to l-asparagine or glutamine; Isoleucine is to leucine or Xie Ansuan; Leucine is to Xie Ansuan or Isoleucine; Methionin is to arginine, glutamine or L-glutamic acid; Methionine(Met) is to leucine or Isoleucine; Phenylalanine is to tyrosine, leucine or methionine(Met); Serine is to Threonine; Threonine is to Serine; Tryptophane is to tyrosine; Tyrosine is to tryptophane or phenylalanine; Arrive Isoleucine or leucine with Xie Ansuan.

When making these changes, the amino acid pro aqua index can be considered.Its wetting ability of every seed amino acid and charge characteristic are assigned to a hydrophilic index, and they are Isoleucine (+4.5); Xie Ansuan (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Halfcystine/Gelucystine (+2.5); Methionine(Met) (+1.9); L-Ala (+1.8); Glycine (0.4); Threonine (0.7); Serine (0.8); Tryptophane (0.9); Tyrosine (1.3); Proline(Pro) (1.6); Histidine (3.2); L-glutamic acid (3.5); Glutamine (3.5); Aspartic acid (3.5); L-asparagine (3.5); Methionin (3.9); And arginine (4.5).

Aspect in giving the protein interaction biological function, hydrophilic amino acid exponential importance is (the Kyte ﹠amp that is commonly understood in the art that; Doolittle 1982, and this paper is cited as reference).Known some amino acid can be replaced by other amino acid that similar hydrophilic index or value are arranged and biologic activity like the reserved category.In the change that produces based on described hydrophilic index, it is preferred that the amino acid pro aqua index is replaced with interior amino acid ± 2, ± 1 with interior be particularly preferred, and ± 5 with interior hydrophilic index or even more particularly preferred.

What this area will also be understood that is that similar amino acid is replaced and can effectively be carried out on the wetting ability basis.United States Patent (USP) 4,554,101 (this paper is cited as reference) have been stated proteinic maximum local average wetting ability, by its contiguous amino acid whose control, interrelated with its immunogenicity and antigenicity, promptly related with the proteinic biological property of the short part of using this non-immunology material.Will be understood that a seed amino acid can be had the amino acid of similar hydrophilicity value to replace by another kind and obtains a kind of biology equivalence, and the protein of immunology equivalence particularly.

As United States Patent (USP) 4,554, describe in detail in 101, below hydrophilicity value be assigned to amino-acid residue arginine (+3.0); Methionin (+3.0); Aspartic acid (+3.0 ± 1); L-glutamic acid (+3.0 ± 1); Serine (+0.3) l-asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline(Pro) (0.5 ± 1); L-Ala (0.5); Histidine (0.5); Halfcystine (1.0); Methionine(Met) (1.3); Xie Ansuan (1.5); Leucine (1.8); Isoleucine (1.8); Tyrosine (2.3); Phenylalanine (2.5); Tryptophane (3.4).

In the change of making based on similar hydrophilicity value, it is preferred that the amino acid pro water number is replaced with interior amino acid ± 2, the amino acid pro water number ± 1 with interior be particularly preferred, and the amino acid pro water number ± 0.5 with interior or even more particularly preferred.

When discussion concentrates on the function equivalence polypeptide that amino acid whose change produces, should recognize that these changes can be subjected to the influencing of change of coding DNA; Consider that also described genetic code is the same amino acid of codon codifieds degeneracy and two or more.The table that provides amino acid and codon thereof in this article is for (as designing probe and primer etc.) use in this type of embodiment and in other purposes.

Following embodiment is included to prove the preferred embodiments of the invention.One of ordinary skill in the art would recognize that the disclosed in an embodiment technology that it is followed is represented the disclosed technology of inventor function well in the present invention's practice, thereby can be considered to the preference pattern of its practice.Yet, the disclosure, those skilled in the art will be understood that and can carry out many changes and obtain similar results and do not depart from aim of the present invention and scope in the specific embodiments that is disclosed.

Embodiment 1

Genitaloid cultivation

A. the preparation of feeder layer

STO cell (Ware and Axelrad, 1972) is cultured in and replenishes with the 2mM glutamine, 15% foetal calf serum (selected batch, Summit Biotechnology), the improved Eagle substratum of Dulbecco of 0.1mM β mercaptoethanol (PEG media).When cell was in the logarithmic phase growth, by tryptic digestion, centrifugal collection was resuspended in 50 milliliters of test tubes, and its concentration is no more than every milliliter 100 ten thousand and accepts 3.2 kilorad cobalt irradiations with deactivation.

After the deactivation, cell is being wrapped in the tissue culturing plate of quilt with 0.1% pig gelatin in advance by the density plating with per 35 millimeters holes 2.5 hundred ten thousand.It is crucial that this density also has been determined separating pig and ox EG clone than the high several times of the employed density of mouse ES cells.After the overnight incubation, described feeder layer is standby.

1. feeder layer density analysis

For quantitatively feeder cell density is to the influence of pig and the cultivation of ox primordial germ cells, pig and ox primordial germ cells are cultured on the feeder cell with three kinds of different densities.

Separate sex-ridge and the basic as following ox tire that separates 3-5 centimetre of crown rump length from 25-27 days pig tires.PGCs with number is on the feeder layer of per 35 millimeters holes 0.5,1.5 and 3.0 hundred ten thousand in STO density by plating.Cultivate after 6-9 days fixed cell colony in 4% formaldehyde, alkaline phosphatase staining, and counting.In addition, write down the form of described colony by photomicrography.Alkaline phosphatase (AP) is active to be determined as the description of Moore and Piedrahita (1997) substantially.Culture plate simply wash twice with phosphate buffered saline (PBS) and in 4% formaldehyde PBS solution room temperature fix 15 minutes.The fixed cell is washed twice with PBS and naphthols AS-MX phosphoric acid salt (200 mcg/ml in 100mM Tris damping fluid; Sigma) and Fast Red TR salt (1 mg/ml, Sigma) in, pH8.2, room temperature dyeing 15 minutes.Stop staining reaction by in PBS, washing culture.The dyeing specificity is passed through at tetramisole (500 μ M; Sigma) under (a kind of AP inhibitor) exists some hole is dyeed to determine.

Plating has epithelium sample morphology than ox on the low density feeder cell and pig PGCs, even and keep an active word of AP also seldom.By contrast, the colony in high-density rearing's cell has morphology and the high AP activity relevant with the EG cell with not breaking up ES closely.In addition, can see typical maxicell nuclear and the outstanding kernel relevant with the EG cell with ES.The number of cell colony also is subjected to the influence of described feeder layer density.Table 7 shows the influence (experiment in triplicate to carry out) of feeder layer density to the pig primordial germ cells behavior of cultivation, and table 8 shows the ox genitaloid influence (experiment in triplicate to carry out) of feeder cell density to cultivating.Only carry out the counting of alkaline phosphatase positive cell colony.

Table 7

(X 10 for density ⁶)	?R1	?R2	?R3	Average
(X 10 for density ⁶)	?R1	?R2	?R3	Average	?3.0	?402	?410	?526	?446
?1.5	?305	?276	?390	?324	?3.0	?402	?410	?526	?446
?1.5	?305	?276	?390	?324	?0.5	?114	?88	?123	?108

Table 8

Density (1,000,000)	????R1	????R2	Average
Density (1,000,000)	????R1	????R2	Average	????3.0	????42	????36	????39
????1.5	????23	????41	????32	????3.0	????42	????36	????39
????1.5	????23	????41	????32	????0.5	????6	????12	????9

As previously noted, increase feeder cell density there is wholesome effect in PGCs with undifferentiated state propagation in cultivation.Enter the substratum except that described STO cell may discharge somatomedin, it is that the undifferentiated morphology of dimension is needed that obvious PGC directly is attached to described STO cell.When appointing, PBC is in sight to be grown directly upon on the plastics, even when being surrounded by the STO cell, it will be grown and propagation is an epithelial lining.Therefore, the extracellular matrix components of being supplied with by the STO cell probably is that the PGC derived cell duplicates needed with a kind of undifferentiated state.In addition, the inventor adds 1-1.5 * 10 to show per 3 to 5 days ⁶Fresh feeder cell improve the performance of described PGC cell.

2. the pig embryo fibroblast is to the feeder layer Effect on Performance

For detecting when using off-gauge STO feeder cell, whether PGC colony number can increase, and has detected the service condition of pig embryo fibroblast (PEF).For separating described PEF, obtained the 25th day instar embryo, remove all inside internal organs and head, and the chopping remaining tissue.Then described tissue temperature was incubated in trypsinase 10-20 minute, and the cell suspension that obtains by plating in the PES of no somatomedin substratum.Every 2-3 days passage cell is once and only for using for two weeks.

With regard to the experiment of feeder cell, be identical to the processing of the processing of PEF cell and STO cell.Described cell tryptic digestion, collection is also passed through 3-10 kilorad radiation deactivation.After the radiation, with per 35 millimeters plate 0.5-3,1,000,000 cell density plating feeder cell on the gelatinization flat board.

New PGC ' the s that collects is by among the STO or STO+PEF (50 50 ratio) feeder cell of plating in the PES of somatomedin is arranged.Check cell colony after electroporation 10-12 days.Colony number in described STO-PES hole surpasses the twice (325 to 128) in independent STO.And, big also occur very fast of described colony.Use the experiment of PEF feeder cell in progress separately.Yet the inventor expects to use described PEF " to stablize " described STO feeding system, and makes its easier enforcement.

3. Prostatropin is to the feeder layer Effect on Performance

The STO feeder cell were by plating in independent PES substratum continuous 48 hours or containing the continuous same time in the PES substratum of 40 nanograms/milliliter bFGF.Having measured bFGF is the isolating essential compositions of pig primordial germ cells.Do not understand still at present whether bFGF directly works to the multiplication rate of described PGCs or described influence whether the change by described STO cell physiological influence PGCs then and work.Since proved already might no solubility STEM CELL FACTOR with (or) separate PGC derived cell colony during leukaemia inhibitory factor, therefore, perhaps bFGF plays PGCs proliferation-inducing thing and differentiation inhibition dual function, or more likely bFGF stimulates the generation of STO feeder cell to separate relevant somatomedin with described PGCs.Visible significant form changes in feeder cell, particularly sees fiber-like and arrange when bFGF exists.

4. extracellular matrix/matrix dirt settling is to the participation of differentiation

Extracellular matrix involves (Meredith etc., 1993) mutually with apoptosis.Extracellular matrix with (or) the matrix dirt settling may participate in the prompting that ES and EG differentiation obtains three different researchs.At first, in the system of no feeder cell, this system comprises the tissue culturing plate with 0.1% gelatin bag quilt to mouse cell by plating.When adding LIF, described cell well-grown, but be not optimal.The described cell of tryptic digestion and being divided into two then, half new flat board that goes down to posterity, and second half is put back into old flat board.The cell growth of returning old flat board is fine, and described cell is mostly to be undifferentiated and healthy outward appearance is arranged.Initiating cell may stay some trypsinase and not remove or remove incomplete material.

The second, unless cultivate altogether with mouse ES cells, rat ES cell never is separated.It is all invalid to cultivate rat ES cell on different feeder cell.The working conditions substratum is also invalid.This prompting mouse ES cells is secreted some material makes rat ES cell breed with undifferentiated state.

The 3rd, when using the low density feeder cell to cultivate pig PGCs, the growth of only a few colony is arranged, and a few cell of described growth is always with among described feeder cell contact directly.Even on described plastics, do not had the colony growth by the encirclement of STO cell.Therefore, the unlikely generation soluble factor of described STO ' s.In addition, for separating pig PGC derived cell, absolute demand adds the bFGF factor to feeder cell.Described bFGF significantly changes the form of described feeder cell.Therefore, compare with directly the PGC cell being worked, bFGF can work with the generation of promotion/inhibition matrix components to feeder cell.And, when during with interior feeder cell upper flat plate inoculation pig PGCs, obtaining the cell colony of only a few preparation 6 hours.Yet the feeder cell of plating preparation in preceding 24 hours produce much better result.Therefore, may be that some matrix components is synthetic and secrete needed during this period of time.

Extracellular matrix with (or) effect of matrix dirt settling in ES and EG differentiation study with many diverse ways at present.At first, plating ES cell shifts out described cell with tryptic digestion, adds pig PGC derived cell then.Secondly, PGCs by plating in matrigel.Its three, PGCs by plating in the actual substrate composition.At last, healthy feeder cell are " extracted ", kill described cell thus and stay matrix, then plating PGCs with contrast corresponding " being extracted " thing of living in.

B. pig, ox and and the generation of billy goat PGC (EG) cell

1. pig and ox PGCs

35-40 days the cattle uterus and the ovary of the 25th day pig uterus of gestation and ovary and gestation are collected when performing the operation or butchering.Each embryo in the gravid uterus shifts out and is placed in the trapping medium by operation under aseptic condition, comprising the PBS that contains 0.4% BSA and 1% penicillin-Streptomycin sulphate level (Gibco#15140-015; 100X:10, the penicillin of 000 unit, the Streptomycin sulphate of 10,000 micrograms).After the collection, described embryo is rinsed in trapping medium twice and dissects respectively under stereoscopic microscope.Identify the sex-ridge of embryo in the growth and under the assistance of tweezers light and slow dissection it.Isolated sex-ridge is placed in the trapping medium and is dissected up to whole embryos.Incubate described sex-ridge 20 minutes and dissociate PGC ' s dissecting medium (0.02% EDTA, 0.8% NaCl, 0.02% KCl, 0.115% hypophosphite monohydrate sodium, 0.02% potassium primary phosphate, 0.02% glucose and 0.001% phenol red) temperature then from sex-ridge.After temperature is incubated,, push the described PGCs of light and slow release in substratum by cover tweezers with the broken ridge of the acupuncture of No. 27 specifications.Described PGCs is collected in the improved Eagle substratum of 3-5 milliliter Dulbecco Ham F10, be supplemented with the 0.01mM non-essential amino acid, 2mM L glutamine, 15% foetal calf serum (selected lot number, Summit Biology) and 0.1mM β mercaptoethanol (PESmedia).Remaining tissue is by the light and slow destruction of suction pipe, and the cell suspension that produces was with 250g rotation 5 minutes.Described supernatant is removed, and 1, centrifugal 5 minutes of 500g.

The throw out that contains PGCs is resuspended in the PES medium.After the collection, described cell by centrifugal wash 3 times and be resuspended in contain 30 nanograms/milliliter soluble recombined human STEM CELL FACTOR, in the PEG substratum of 40 nanograms/milliliter Prostatropins and 20 nanograms/milliliter LIF and plating on the STO feeder layer.In addition, when having or not having STEM CELL FACTOR and exist, measure and add the effect of 100 nanograms/milliliter uterus ferritins alkaline phosphatase enzyme positive colony number.

Density is 10, the described cell suspension of 000PGCs/ milliliter by plating on STO cell feeder layer.After cultivating in 10-14 days, there is the morphologic colony of ES sample to be gone down to posterity fresh feeder layer to set up clone.The colony that produces by tryptic digestion with the every 6-9 days new feeder layers that go down to posterity at interval.The differentiation state of separated cell is determined (Talbot etc., 1993) by morphology and alkaline phosphatase expression of enzymes (a kind of mark of not differentiating embryonic cell).

Feeder cell perhaps are compositions the most key in the total system.In order to obtain most probable number MPN purpose EG colony, the density that needs is per 35 millimeters culture dish 2.5 hundred ten thousand.These are different with mouse ES cells, and its required density only is per 35 millimeters culture dish 100 ten thousand.And the trial of cultivating the EG cell on freshly prepd feeder layer all fails.And obtained optimum when the feeder cell bed board re-uses after 24 hours at least.

2. billy goat PGCs

Obtain one single 25 day age the goat tire and as above method separate sex-ridge.Choose separation method with the primary pin and separate PGCs and plating on the STO feeder layer, contain uterus ferritin rose and LIF in its substratum, SCF and bFGF, concentration is as mentioned above.The colony that obtains was protected for two generations, and cell is fixed and does alkaline phosphatase activities dyeing during this period.

Billy goat PGC that cultivates and pig and ox cell finding morphology are basic identical.And described billy goat cell dyeing is the strong basicity phosphatase activity.The morphology of billy goat PGCs and AP dyeing mode class are similar to the pig of as above demonstration and the pattern of ox PGCs.The colony of describing cultivation as this paper goes down to posterity twice before record morphology and alkaline phosphatase activities at least.In all cases, all observe minicell matter, maxicell nuclear and the typical ES sample morphology of giving prominence to kernel.In addition, the colony that does not adsorb after the guarantor is gone down to posterity for the first time demonstrates the ability (following embodiment 2) that is divided into simple idiosome.In caprine situation, after the colony of guarantor suspension normally goes down to posterity new feeder layer, grow and saw described idiosome in 4 days.In all cases, the typical double layerization of simple idiosome is apparent is significantly and has shown the versatility feature of described isolated cell system.

Pig, ox shows that with the behavior similarity of billy goat PGC when plating is in culture systems described herein this system might be common to many species separates PGC derived cell system.It is also important that it is shown in the progress that forms in the species in advance and can be easy to be applied to another species.At present, inventor's separation that just beginning one's study, genetic transformation and detect the versatility of goat PGC derived cell system.

3. the PGCs of other species

Whether can be used to separate the EG cell of other mammalian species for measuring present method, the rabbit from cultivate separates PGC and measures the ability that it develops into the PGC derived cell colony of tool ES sample form with rat.In brief, separate PGCs from 15-18 days New Zealand white rabbit tires with 11-12 days Sprague-Dawly rat tires.9.5-12.5 the mouse tire of it gestation is used as contrast.The PGC separation steps is to shift out sex-ridge and the described tissue of tryptic digestion, and plating is to the STO feeder layer of deactivation then.Substratum is made up of Dulbecco improvement Eagle substratum Ham ' s F10 substratum, replenish with the 0.01mM non-essential amino acid 2mM glutamine, 15% foetal calf serum (selected lot number), 0.1mM 2 mercapto ethanol and human stem cell factor, rh-bFGF and people LIF.With regard to the cell dimension, the colony that goes down to posterity was to new feeder cell in every 8-10 days.

The gained colony is by its morphology, and alkaline phosphatase (AP) dyes, and the ability of vitro differentiation is identified.In all situations, all obtaining has the morphologic colony of EG sample, although slightly different between the species, round and more be " grape sample " as the mouse colony than other species.Equally, though the AP activity is all having discovery in the species, strength of signal sharply descends in rabbit.The described PGC derived cell of these results suggest can be from rabbit, separates with rats in vitro and cultivates, and provides thus to analyze its developmental potentiality and they on the accurate basis of the purposes of these species of genetic modification.

C. alpha2 Macroglobulin is to the influence of PGC culture

Add uterus ferritin (Utr) and demonstrate the increase of the PGC colony number of gained in the described substratum.When only adding the uterus ferritin when causing some to promote to described substratum, determining the most effective molecule is not independent uterus ferritin, but is attached to the uterus ferritin (serpin of serpin; Malathy etc., 1990).For measuring the beneficial effect that serpin itself is provided, studied the proteinase inhibitor molecule that is called alpha2-macroglobulin (Feige etc., 1996) of wide scope.The wide scope proteolytic enzyme of the known irreversible inhibition of this molecule (Feige etc., 1996), and in conjunction with and transport cytokine.

Be not subjected to the constraint of any specific explanations, the binding mode of alpha2-macroglobulin is considered to work by the necrocytosis (PCD or apoptosis) that suppresses sequencing in PGC.The necrocytosis of sequencing is normally (among the embryo; Pesce and Felici, 1994) all existing record and among the PGCs (Pesce etc., 1993) that cultivates.Serpin has involved the restraining effect (Tewari and Dixit, 1995) of PCD.

As mentioned above, the method for collecting PGCs from the tire of growing is included in pre-the cultivation 20 minutes the EDTA/ glucose solution, chooses sex-ridge with pin then and described PGCs is oozed out.The inventor determines to study oozes soft organizing whether to have and being better than pre-the cultivation and advantage that pin is chosen of just having collected, and whether alpha2-macroglobulin has any benefit to two methods one of any.

Collect sex-ridge from 25-27 days pig tires, be divided into four groups (experiments of a kind of 2 * 2), be positioned over or alpha2-macroglobulin 1 mcg/ml (two groups) is arranged or do not have in the PES substratum of alpha2-macroglobulin (two groups), and pin is chosen so that cell colony oozes out (two groups) or ooze soft (two groups) under the assistance of tweezers immediately.In brief, described ridge makes it morsel by pushing them with the back side of tweezers to the plate bottom.Described ruined tissue is by making its pin that passes No. 20 specifications destroyed and further decomposed up to whole tissue block for several times.After disorganization, with the centrifugal sample 3-5 of 250 * g minute with sedimentation tissue fragment and collect that to contain major part be single celled supernatant liquor, and centrifugal 5 minutes at 1000 * g.The gained throw out is resuspended in and contains LIF, SCF and bFGF, have or the PES of unmanned alpha2-macroglobulin in, carry out cell counting and triplicate plating in feeder layer.

After six days, fixed cell colony, alkaline phosphatase staining, and counting.Table 9 provides described separation method and adds alpha2-macroglobulin develops into the alkaline phosphatase activities colony number of cell to PGCs to described substratum influence.

Table 9

Pig #	Separation method	Substratum	?R1	?R2	?R3	Inoculating cell	Standardized value ^*
Pig #	Separation method	Substratum	?R1	?R2	?R3	Inoculating cell	Standardized value ^*	?1	Pin is chosen method	PES	?2608	?3640	?2936	?247	?12399
?1	Ooze soft method	PES	?4216	?3856	?3976	?371	?10842	?1	Pin is chosen method	PES	?2608	?3640	?2936	?247	?12399
?1	Ooze soft method	PES	?4216	?3856	?3976	?371	?10842	?1	Pin is chosen method	MAC	?4688	?4960	?5424	?277	?18087
?1	Ooze soft method	MAC	?4712	?5176	?4368	?290	?16395	?1	Pin is chosen method	MAC	?4688	?4960	?5424	?277	?18087
?1	Ooze soft method	MAC	?4712	?5176	?4368	?290	?16395	?2	Pin is chosen method	PES	?3904	?3776	?5000	?263	?16104
?2	Ooze soft method	PES	?3368	?3488	?2840	?380	?8532	?2	Pin is chosen method	PES	?3904	?3776	?5000	?263	?16104
?2	Ooze soft method	PES	?3368	?3488	?2840	?380	?8532	?2	Pin is chosen method	MAC	?6200	?6008	?6864	?170	?37380
?2	Ooze soft method	MAC	?8312	?7872	?8896	?233	?35865	?2	Pin is chosen method	MAC	?6200	?6008	?6864	?170	?37380

^*Standardized value is represented as colony number of cell in per 1,000,000 cells

Obviously, alpha2-macroglobulin has positive influence to the ability that PGC breeds in cultivation.As if described influence bigger when choosing the systematic collection cell by violent crushing system than by more light and slow pin.May be that violent gathering system causes more inducing the proteolytic enzyme of programmed cell death to discharge, and alpha2-macroglobulin cause high-level provide protection with combining of these proteolytic enzyme.On the contrary, more light and slow pin is chosen system induces lower level in described separation method PCD, and thereby is the corresponding lower reason of provide protection of alpha2-macroglobulin.Therefore difference between two pigs (being that pig shows bigger reactions No. 2) is attributable to time between collect in the uterus and sexual cell separates longly makes the action time of the factor pair PGCs that is discharged by thanatogenic tissue longer.Regardless of reaction mechanism, be not difficult to find out that from above-mentioned data adding alpha2 Macroglobulin has positive influence to described substratum to the number that alkaline phosphatase positive cell in the training period occurs.

Alpha2-macroglobulin is also studied the influence of passage cell.The ability that normal observed alpha2-macroglobulin prevents loss when detecting PGCs when cultivation by tryptic digestion, cell is gone down to posterity twice having or do not have in the presence of the alpha2-macroglobulin, described cell colony is fixed, and alkaline phosphatase staining is also measured the number of the positive colony of alkaline phosphatase staining.PGCs is collected among the PES and plating is having or do not having on the feeder layer of the substratum that contains 1 mcg/ml alpha2-macroglobulin.Made cell colony growth 7 days, as above describe carry out tryptic digestion and once more plating having or do not having in the substratum of alpha2-macroglobulin.Sample is duplicate, and revises the cell number that is added into.

The following first-generation PES MAC of experimental design s-generation PES MAC PES MAC

The adding alpha2-macroglobulin has increased the number (table 10) of first-generation colony to described substratum.In addition, no matter whether described cell before once (+) being arranged or not having the cultivation down of (-) this molecule (table 11), all has the protective effect of alpha2-macroglobulin to the s-generation.As if therefore, add alpha2-macroglobulin or other proteolytic enzyme or apoptosis supressor and will help the PGCs initial gross separation colony cultivated to described substratum, and it is also important that, it can increase the efficient of setting up EG clone steady in a long-term.

Table 10

Substratum	?R1	?R2	The cell (k) of inoculation	Standardized value ^*
Substratum	?R1	?R2	The cell (k) of inoculation	Standardized value ^*	PES	?4728	?1680	?433	?7401
MAC	?8000	?6304	?325	?22028	PES	?4728	?1680	?433	?7401

^*Standardized value is represented as colony number of cell in per 1,000,000 cells.

Table 11

The substratum first-generation	The substratum s-generation	R1	?R2	The inoculation colony	Standardized value ^*
The substratum first-generation	The substratum s-generation	R1	?R2	The inoculation colony	Standardized value ^*	+	+	3680	?3920	?1850	?2052
+	-	2120	?3480	?1850	?1512	+	+	3680	?3920	?1850	?2052
+	-	2120	?3480	?1850	?1512	-	+	1000	?1277	?801	?1422
-	-	877	?857	?801	?1084	-	+	1000	?1277	?801	?1422

^*Standardized value is represented as colony number of cell in per 1000 plating colony number of cell.

When having or not having alpha2-macroglobulin and exist, cultivate or among the cell colony that goes down to posterity now studying with the ability in undifferentiated state survival number generation.In fundamental research, the colony that obtains from the third generation ox PGC ' s that cultivates the substratum of alpha2 Macroglobulin is arranged is the twice (22 to 45) that does not have to obtain in the substratum of described proteinase inhibitor number.And described colony occurred average early 2-3 days and when cultivating bigger form was arranged when having alpha2-macroglobulin to exist.

Further study to determine the optimum concn of alpha2-macroglobulin.The positive colony ratio of AP is determined in three experiments compared with the control in the sample that contains 1 mcg/ml or 0.5 mcg/ml.Ratio during 1 mcg/ml is 0.81 ± 0.37,0.84 ± 0.23 and 0.83 ± 0.27, and ratio is 1.98 ± 0.36 when 0.5 mcg/ml, 1.43 ± 0.27 and 1.70 ± 0.41.Therefore, than as if in producing the positive colony of AP effectively nearly 2 times of the alpha2-macroglobulins of low dosage.

Embodiment 2

The detection of the PGCs that cultivates

A. pig PGCs

Pig PGCs is contained Prostatropin (bFGF) by plating in the 10 millimeters holes neutralization that contains the STO feeder cell, leukaemia inhibitory factor (LIF), and or the substratum of solubility STEM CELL FACTOR (SCF) or pig uterus ferritin (pUTE) in.Totally 4 groups of parallel laboratory tests.After 10-12 days, carry out alkaline phosphatase staining and counting.Only there are ES sample morphology and alkaline phosphatase male colony to be included in the described ultimate analysis.Following (Donovan etc., 1986 are carried out in alkaline phosphatase staining; Talbot etc., 1993b; Moore and Piedrahita, 1996).Described colony washes twice in PBS, room temperature is fixed 15 minutes in 4% formaldehyde PBS solution, washes 3 times in PBS then.Then in naphthols AS-MX phosphoric acid salt (200 mcg/ml; Sigma) and fast red TR salt (1 mg/ml; Sigma) dye described colony 20 minutes of room temperature in the 100mM Tris buffered soln (pH 8.2).Stop dyeing by washing in PBS.

With regard to each quadruplicate experiment, among the pUTE among colony and the SCF ratio of colony be respectively 5.0,3.0,3.6,2.0.Per 10 millimeters hole colony mean numbers are 4 in the SCF substratum, and are 18 in pUTF handles.And, all there is not influence to the whole morphology of described colony with to the intensity of alkaline phosphatase staining.

B. ox PGCs

Collect two tires in the slaughterhouse and determine gestation between 30-40 days by crown rump length, processing as mentioned above.Isolating ox PGCs is resuspended in and is supplemented with bFGF, in the PES substratum of LIF and solubility SCF, and in the cultivation of the top of the STO of deactivation feeder layer.The gained colony was checked every 2-3 days up to having detected the morphologic colony of suitable EG sample (closely outward appearance and maxicell nuclear and tenuigenin ratio).After cultivating about 30 days, the expression pattern of described EG sample colony is measured in selected hole by alkaline phosphatase staining.When similar colony during, measure the low-level of the epithelium sample colony of expression scope from the high level of highly dense colony to expansion by alkaline phosphatase staining.

C. pig, ox and billy goat PGCs are to the differentiation of idiosome

One of the index that ES and EG cell keep the ability of its versatility feature is their vitro differentiation abilities when being inoculated in the suspension culture base.Described differentiation is fairly individual and generation is called as simple and cryptomere idiosome.For determining that the inventor is from pig, whether goat and ox isolated cells can form idiosome, light and slow tryptic digestion colony is so that shift out and centrifugal described cell mass and be resuspended in no somatomedin and with the conventional ES substratum of calf serum replacement FBS (Piedrahita etc., 1992) from described tissue culturing plate.Described cell mass by plating in non-adsorptivity bacteriology culture plate and 39 ℃ of cultivations.Change substratum every day and write down the morphology that breaks up colony with 24 hours intervals.

Show the ability that forms simple idiosome when all these species are in being inoculated in suspension culture.In caprine situation, after going down to posterity new feeder layer, remaining colony from suspension culture sees the idiosome of growing 4 days.In all situations, the simple typical double-deck outward appearance of idiosome is significantly and shows the versatility feature that described isolated cell is.In the situation of ox, the cryptomere idiosome is observed the bottom attached to described flat board.Though just in this point, described idiosome feature does not exceed the morphological change of record as yet, it has been strengthened when inoculating on suitable culture condition, and separated cytodifferentiation becomes the ability of few kinds of tissues type.

Embodiment 3

The derive electroporation analysis of colony of primordial germ cells

Collect PGC cell (50,000-500,000) immediately it is resuspended in (0.4 centimetre of clearance distance, goods Article Number 165-2088) electroporation in the 0.8 milliliter of PES substratum that contains 5nM linearization plasmid DNA and in the BioRad electroporation test tube under different voltages and impedance conditions after.(GFP, Clontech) plasmid of Zu Chenging and the cytomegalovirus among the pcDNAIII (CMV) promotor (Invitrogen) are joined by the egfp of " humanization ".Described test tube is placed in the BioRad gene pulse instrument of being furnished with capacitance increase electroporation device and electroporation under 300 to 400 volts of voltage ranges and 250 to 500 microfarad capacitance ranges.Behind the electroporation, the resuspension cell is in substratum and with 10, and the density plating of 000PGC/ milliliter was cultivated 8-12 days on the new feeder layer of the STO of deactivation cell.There is the morphologic colony of ES sample to be gone down to posterity new feeder layer to set up clone.The gained colony every 6-9 days by the tryptic digestion new feeder layer that goes down to posterity.The differentiation state of separated clone is measured by morphology and alkaline phosphatase expression of enzymes.

Behind the electroporation 14 days, detect by green fluorescent protein (GFP) and to identify the transgenosis colony.GFP detects by placing cell observation in an inverted fluorescent microscope and under UV-light (FITC filter) and finishes.Under these conditions, when not detecting the non-transgenic colony, seen transgenic cell is green.Record fluorescence colony number.In some flat board, colony has also carried out alkaline phosphatase staining with proof alkaline phosphatase and GFP coexpression.In all the other flat boards, choose the fluorescence colony and the new feeder cell that go down to posterity to set up transgenosis EG clone.

Except that transforming former generation PGC (not cultivating), the colony that goes down to posterity after 1 and 2 time is transformed.Collect on non-transgenic PGCs and the STO feeder layer of plating in containing the PES substratum of somatomedin.10 to the 14 days ES sample colonies in inoculation back were digested 5 minutes and were resuspended in the PES substratum for 37 ℃ by trypsinase in 0.25% trypsinase.100-5000 PGC derive colony with the GFP construction by electroporation as described before, and on the new feeder cell of plating in containing the PES substratum of somatomedin.As above described evaluation and chosen the new feeder layer that goes down to posterity after 10-14 days by the transgenosis colony that these electroporations produce.By use mouthful operation glass pipette from described feeder layer mechanicalness shift out the fluorescence colony finish picking go down to posterity and place described colony 0.25% trypsinase 5 minutes to carry out cell dissociation.Make colony be broken into the little group of cell and unicellular then, and on the new feeder layer among the PES that contains somatomedin that goes down to posterity to set up clone.

A. the influence of electroporation conditions change

About 200,000 new isolating PGCs are divided into four groups, and at 300 or 400 volts and 250 or 500 microfarad electroporations.The cell of every kind of processing is dispensed in two 35 millimeters holes and cultivated 12 days on feeder cell, at this moment, measures the positive colony number (table 12) of alkaline phosphatase enzyme positive and GFP.

Table 12

Handle	The transgenosis colony	The alkaline phosphatase colony
Handle	The transgenosis colony	The alkaline phosphatase colony	300 volts, 250 microfarads-A	?6	?320
300 volts, 250 microfarads-B	?13	?552	300 volts, 250 microfarads-A	?6	?320
300 volts, 250 microfarads-B	?13	?552	300 volts, 500 microfarads-A	?13	?1200
300 volts, 500 microfarads-B	?22	?1060	300 volts, 500 microfarads-A	?13	?1200
300 volts, 500 microfarads-B	?22	?1060	400 volts, 250 microfarads-A	?19	?960
400 volts, 250 microfarads-B	?26	?1072	400 volts, 250 microfarads-A	?19	?960
400 volts, 250 microfarads-B	?26	?1072	400 volts, 500 microfarads-A	?25	?812
400 volts, 500 microfarads-B	?28	?896	400 volts, 500 microfarads-A	?25	?812

B. the genetic transformation of former generation pig PGCs

As above-mentioned isolating PGCs 300 volts and 250 microfarads with 5nM GFP-CVM plasmid electroporation and plating on the STO feeder cell in the mSCF substratum.Behind the electroporation 10 to 12 days, under ultraviolet lamp, observe colony to determine the expressing proteinic colony number of GFP, described protein expression is represented the genetic transformation of plasmid.As a result, colony is carried out alkaline phosphatase staining (a kind of mark of primordial germ cells and undifferentiated state embryonic cell) and counting.Obtained 6221 alkaline phosphatase enzyme positive colonies in the cell of electroporation from 300,000.Wherein, 28 also is that GFP expresses the positive, shows that they are genetically modified.The morphology that is transformed the PGC-derived cell is different from seen in mouse ES and EG colony.In some cases, only the part of a colony is expressed GFP, shows that described colony most probable derives from several cells, wherein has only one by genetic modification.Yet expression of ALP can not be distinguished the transgenosis and the non-transgenic composition of these colonies, shows that described genetic transformation does not disturb the normal physiological function of described PGC cell.

Its intensity of colony that transforms with the CMV-GFP construction is from by force to being difficult to identification.Initial this heterogeneity is owing to the position effect that is caused by described genetically modified insertion at random.Yet, being clear that those have the cell colony that related Morphological is arranged with ES and EG cell, its fluorescence intensity is minimum.On the contrary, inoblast and epithelium sample colony have dense dyeing.This effect is construed as the differentiated result on the described CMV promoter activity.Be difficult to distinguish with differentiation entoderm seen in differentiation mouse ES and EG cell on the outer shroud morphology of GFP express cell.This prompting is in other non--GFP express cell (inner undifferentiated cell), and promotor is activated at differentiation phase.This shortage of expressing not always the fact of this situation most likely because position effect and the differentiation synthesis result in period.This points out described CMV promotor may not be to detect optimum start-up of transgenosis PGCs.

C. the conversion of the first and second generation pig PGC ' s

The PGC of zero generation and the first-generation derives colony by tryptic digestion, and at 400 volts, 250 microfarad electroporations, and inoculation is as above described.Cultivate after 12 days, counting fluorescence colony, and select representative sample further to go down to posterity by going down to posterity respectively.On average detect 40 colonies from each conversion.The initial number of PGC is difficult to calculate, because exist the pollution that tryptic digestion is collected remaining STO behind the PGCs.

From this type of non-former generation conversion, the inventor has the positive colony of above GFP of several two generations of surviving.The amplification of these clones is carried out DNA extraction and analysis.

S-generation pig PGC colony and the former generation PGC colony of deriving of deriving has similar morphology.Identified and dissociated 5 minutes by the picking passage cell with in 0.25% trypsinase and break away from the colony of expressing described GFP after the described CMV-GFP construction electroporation with described feeder layer.After dissociating, colony is placed on the new feeder cell in the 24 hole flat boards and cultivates.Shifted back 10 days, and can see a plurality of colonies of appearance in each flat board.The morphology of colony and former generation PGC derive and can not distinguish seen in the colony.This shows that cultivation that employed culture systems allows described transgenic pig PGC derived cell is through number Dai Erwu differentiation.Say from practical perspective, this means that the former generation colony of might going down to posterity can be used for genetic analysis and is used to produce chimeric amount of material with increase.In other words, some colony seen in the s-generation can be collected and pass through PCR ^TMAnalyze to identify and the corresponding target transgenosis of non-target colony.This just allows that evaluation passed through the pig derived cell of homologous recombination.

The derive back several generations of colony of PGC dissociates with trypsinase, is resuspended in to contain in the 5nM CMV-GFP substratum and at 300 volts and 250 microfarad electroporations.Plating and cultivate 7-10 days after, analyze exist in the described culture the positive transgenosis colony of the morphologic AP of the ES of differentiation sample arranged.Dissociate new feeder layer in transgenosis colony and each 24 hole flat board that goes down to posterity to set up transgenic cell line with trypsinase.The morphology of this s-generation colony and GFP express with former generation colony corresponding aspect do not have difference.Yet, have more GFP on the colony of morphology of differentiation express and receive publicity.This change scope rolls up to expression from the GFP expression decreased.In addition, containing some that express the district with the negative district of GFP GFP arranged side by side mixes colony and is observed.

D. the genetic transformation of ox PGCs

From the ox PGCs of the embryo collection of 4-6 centimetre of crown rump length 400 volts and 250 microfarads the PES substratum by electroporation.After 5nM was same as the CMV-GFP construction electroporation of the structure that above-mentioned pig PGC uses in transforming, cell was on the STO of per 35 millimeters plates 3.0 hundred ten thousand in density by plating and cultivated 6-9 days.From the 6th day, and up to the 9th day, by the fluorescence microscopy colony to identify the positive colony of GFP.

Several colonies are identified expressing green fluorescent protein matter.As use the situation of pig cell, as if the morphology of described transgenosis colony as broad as long with seen in the non-transgenic contrast.Described transgenic cell moves among the ability that produces the tire of can surviving with blastocyst injection studying at present by consideration convey.

Compare with pronucleus injection, the ability of conversion ox PGC cell has represented to cultivate then by the described sexual cell of direct processing and has moved with consideration convey or mosaic formation produces the possibilities of transgenic animal heredity in.PGC has several advantages that exceed the pronucleus injection.At first, before carrying out the versatility detection, pass through PCR ^TMOr the southern blotting technique analysis can determine the sex of donor PGC, allows the transgenic animal sex that prior prophesy produces.Secondly, owing to can obtain several transgenosis colonies from single tire, the tire that might use superior hereditary feature is as the PGC donor, and obtains transgenic cell from this donor.These cells can be used to that consideration convey moves or the pronucleus injection then.Therefore, the gained transgenic animal will not only have required sex but also will have the further improved superior hereditary feature of heredity.And with regard to existing pronucleus injection technique, the needs of injecting thousands of embryos make to use superior hereditary feature (female generation and parent) expense expensive as can't to bear and not practicable in logic.

The 3rd, if moving, consideration convey is used to produce filial generation, needed embryo (tire) donor and receptor number will significantly reduce, only owing to a) only need a tire can obtain hundreds of transgenic cells; And b) have only grow to blastocyst stage and by marker detection with (or) PCR ^TMConfirmation is that genetically modified embryo will be transferred.The ratio of becoming pregnant after promptly using the cultured cells consideration convey to move is low to 10% (Stice etc., 1996), only needs 10 acceptors just can obtain single transgenic animal.At present, before obtaining single transgenic animal, need injection and more than 50 embryos to 250 of transferase 12 acceptor (Wall, 1996).And last, because a transgenosis colony comprises 20-100 consistent cell, might clone the transgenic animal of several basically identicals.Freezing several the gained embryos of this tolerable arrive amortization period and are determined by allowing it up to one of described clone's phenotype.If this phenotype is considered to superior, the consistent animal of number cover can be thawed and be produced to described frozen embryo.

At portion in the recent period in the report, reported that the embryo culture of deriving participates in the ability (Cibelli etc., 1997) that the fetal hair after consideration convey moves is educated.It is relevant with AP expression forfeiture with differentiation that the morphology that described clone is derived and had epithelial types from the inner cell matter of growing embryo, this morphology have been proved to be.And the nuclear energy of these clones enough participates in the growth of tire.Yet, during by the 50th day, caused tire to lose (Stice etc., 1996) from whole gestation of this cell type, show that therefore they can not form the survival offspring.

On the contrary, isolated cells of the present invention has kept the representative configuration of ES cell and EG cell and has expressed AP.Confirm that as previous the ICM derived cell can participate in normal embryo development (Keefer etc., 1994), and because the morphology and the AP activity of this cell are similar to ICM, they after moving, consideration convey should be able to produce the offspring of survival.In addition since with the similarity of above-mentioned pig cell, they should be able to (as pig) participate in mosaic and form.

Embodiment 4

The contribution that the pig PGC that transgenosis is cultivated forms mosaic

Identify separated as mentioned above and by the transgenosis PGC that transformed, dissociate by tryptic digestion and get segmentation cavity or the injection that 10-15 injection cell enter blastocyst in the growth and enter morular closely inside.Shift injected embryo and collected tire in 25 days to the synchronization acceptor and in gestation.Be assessment, under luminescent lamp, observe the embryo to detect the GFP mark.After visual inspection, the chopping embryo also gets one section tissue and is used for DNA analysis.DNA analysis is by passing through PCR ^TMOr the Southern DNA analysis detects described GFP formation.

(Gentra Systems, Minneapolis is MN) by salting-out process (Miller etc., 1988) DNA isolation to use Purgene DNA separating kit.After the separation, DNA is resuspended in the TE damping fluid, and spending the night at 55 ℃ makes its dissolving and detect optimum density to determine concentration.With neo primer or GFP primer through 35 round-robin PCR ^TMCarry out the DNA PCR of 100ng ^TMAnalyze.Fragment is separated by agarose gel electrophoresis, with ethidium bromide staining and photograph.

Analyze enzyme (Boehringer Mannheim, Indiannapolis, IN shown in using about Southern; Promega, Madison, WI; Or New England Biolabs, Beverly, MA) digested genomic dna (5-10 microgram) spends the night, and sample is uploaded on 0.7% sepharose, by separating at TAE damping fluid (0.04M Tris-acetic acid, 0.001M EDTA) electrophoresis.Described DNA by capillary transfer device (Sambrook etc., 1989) be transferred to the N-Hybond film (Amersham, Arlington Heights, IL).Dry then described film is also used ultraviolet light cross-linking before hybridizing with radioactive probe.RapidHyb solution is used in hybridization, and (Amersham, Arlington Heights IL) carry out according to the method for manufacturer.

Follow the method for manufacturer, with [ ³²P]-(Amersham, Buckinhamshire is England) with commercial test kit (High Prime, Boehringer Mannheim, Indianapolis, IN.) the described probe of mark (50 microgram) for dCTP.By the description in manufacturer's method, (Promega, Madison WI) remove uncorporated Nucleotide by making reactant cross Sephadex G-50 post.The probe of heating (95 ℃, 10 minutes) sex change mark is with every milliliter of hybridization solution 10 ⁶Radiolabeled probe of cpm and the hybridization of described film, described reactant temperature on 65 ℃ of shaking tables was incubated 2 hours.After the hybridization, described film is rinsed twice, then in room temperature with 2 * SSC, 1%w/v SDS solution was washed 20 minutes.After washing, filter membrane is placed in the disposable seal type tableware plastics bag (seal-a-meal plastic bag), is sealed in exposure casket (Eastman Kodak company, a Rochester that intensifying screen and the X-OmatAR of Kodak film are arranged, NY.) in, and-70 ℃ of placements 1 to 4 day.

In 7 acceptors accepting the injection embryo, become pregnant for two.Obtain 14 normal tire and from these two gilt and only need molten tire.Then, by the southern blotting technique analysis GFP and neo transgenosis that these tires exist are analyzed.Digest from developmental tire DNA isolation and with HindIII restriction restriction endonuclease.The fragment of described digest is separated by gel electrophoresis and is transferred on the nylon membrane.Seek and visit described film with GFP and neo gene probe then.

In 14 analyzed normal tire, when all hybridizing with GFP and neo, one is that strong positive and one are the weak positives.Be that molten tire separated DNA does not show described genetically modified existence from disappearing unexpectedly.Transgenosis is that two tires of male are selected for further analysis, to get rid of the plasmid contamination of heavy.

The DNA that is separated to from two positive tires with can plasmid pollute with the DNA that is inserted in the genome between make differentiation restriction enzyme digest.Described DNA be not used for the inner cutting of GFP/neo plasmid that described conversion is used with Bst * 1 digestion, this enzyme.Bst * 1 digest shows the band of a 8kb and described GFP and neo probe hybridization.Because initial GFP/neo plasmid construction thing has only 6.2kb,, then can not obtain big hybrid belt unless described construction is to be embedded in the described genomic dna of two Bst * 1 site flank.In addition, this shows to have only a described construction of single copy to be inserted in the described genomic dna.Though can not estimate the contribution ratio of described transgenosis colony to described tire, the transgenosis in one of described tire can be easy to be shown that by the detected fact of genomic dna engram analysis this contribution is tangible.

Aforesaid research is repeated, and obtains three conceived gilt.From 2 gilt, obtain 19 normal tire.GFP and the neo transgenosis that exists as tire as described in the above-mentioned analysis by the southern blotting technique analysis then.One of tire that reclaims is by genomic dna trace and PCR ^TMAnalyze and be proved to be genetically modified.The fluorometric analysis of the sex-ridge of this tire has shown the existence of fluorescence band, shows the expression of egfp mark.The expression of described GFP shows that described tire contains transgenic cell in its sex-ridge.This points out in the sexual cell that described transgenosis can be present in described tire, and therefore to carry out kind be transmission.

Remaining gilt of becoming pregnant is allowed that gestation is to amortization period.Gilt bears three piglets, and one of them proves mosaic by the genomic dna engram analysis.And described transgenosis is present in 70% the tissue (comprising testis and epididymis), and prompting kind of the possibility that system transmits (table 13, as follows).In addition, be incorporated into really in the described karyomit(e), carry out restrictive diges-tion with HindIII and BamHI for confirming described transgenosis.Detect a 6.0kb HindIII fragment and a 9.5kb BamHI fragment.Both sizes show that described detection signal is not that the plasmid pollution causes.

Table 13

The detection of GFP in the stillbirth transgenosis mosaic tissue

Types of organization	??S1*	??S2	??S3	??S4	??S5
Types of organization	??S1*	??S2	??S3	??S4	??S5	Liver	??+	??-	??-	??-	??-
The heart	??++	??+++	??+			Liver	??+	??-	??-	??-	??-
The heart	??++	??+++	??+			Lung	??+/-	??+/-	??+/-	??+/-	??+
Kidney	??+	??++	??+			Lung	??+/-	??+/-	??+/-	??+/-	??+
Kidney	??+	??++	??+			Epididymis	??+++	??+++
Testis	??+	??-				Epididymis	??+++	??+++
Testis	??+	??-				Skin	??-	??-	??+	??++	??-
Muscle	??+	??-	??-	??+	??+	Skin	??-	??-	??+	??++	??-
Muscle	??+	??-	??-	??+	??+	Placenta	??+/-	??+/-	??++
Large intestine	??+	??+				Placenta	??+/-	??+/-	??++
Large intestine	??+	??+				Small intestine	??+	??+/-
Thymus gland	??-	??-				Small intestine	??+	??+/-
Thymus gland	??-	??-				Femur marrow	??-	??+
Spleen	??+	??+/-	??++			Femur marrow	??-	??+
Spleen	??+	??+/-	??++			Pancreas	??+	??+/-
Umbilical cord	??+					Pancreas	??+	??+/-
Umbilical cord	??+					Stomach	??+	??+	??+

^*Sample number.Bigger tissue is according to its size and complexity repeatedly sampling at random.

-, no detection signal; +/-, little detection signal; +, detection signal is arranged; ++, strong detection signal is arranged; +++, has extremely strong detection signal.

Research is in addition carried out with PGCs such as the above-mentioned method of Duroc pig, and described pig has red skin and red hair.The cross-fertilize seed of using is by Yorkshire, Landrace, and the hybridization between the Hampshire and the black and white kind of coming.In case Duroc derived cell system is injected in the hybridization embryo in the hope of mosaic birth, the erythema to a certain degree that it can have proof to have an effect from described injection clone on its skin.The mosaic of two kinds of fur colors is identified.Chimeric the breeding of described fur color will determine whether that the kind system that described PGCs has taken place transmits.

The data that provides above clearlys show that transgenic pig EG cell has the ability the growth of tire is worked when injection enters host's blastocyst.This research also points out described mosaic to have a kind system effect.Therefore, using early stage passage cell is strategy preferred for this invention at present.

Passage number is how to influence described cell mosaic is formed among the ability play a role studying at present.Because the inventor can keep cell to reach 5 months and go down to posterity 10 times and still keep alkaline phosphatase activities and normal morphology, how many pig PGCs versatility characteristic aspect that is expected at cultivation do not have and reduces.Shim etc. (1997) report in the recent period can play a role to mosaic formation after showing non-transgenic PGC derived cell long-term cultivation, and this report has been strengthened these transgenosis colonies and the potentiality of clone aspect the generation transgenic pig effectively.

According to these results, obviously different with the embryo derived cell, PGC derives colony can be with the longer time of undifferentiated state existence (as being measured by morphology and AP dyeing) in cultivation.What is interesting is that the morphology of described EG colony more is similar to morphology (Piedrahita etc., 1990 of mouse ES cells than previous isolating pig ES like cell; Gerfen and Wheeler, 1995).The pig ES like cell of previous report has outward appearance not too closely, also differentiates most of cell easily even be difficult for differentiating whole cells herein.On the contrary, EG cell described herein has outward appearance very closely, is divided into the epithelium individual layer unless be difficult to differentiate the tracked colony of each cell herein.Though this difference may be only owing to the huge difference (wherein the PGC number exceeds thousand times) of the initiator cell number between two kinds of systems, this species diversity may be relevant with the difference in LIF and the CNTF expression of receptor.

Measure feeder cell density and be owing to observe and do not tend to be divided into fast the epithelium individual layer with the colony that feeder layer directly contacts to the research of the influence of colony number.The increase of this prompting feeder cell density (colony that therefore guarantees whole platings be in directly with the contacting of described STO cell in) may be favourable.This paper result supports this observation, but it is also important that, the influence of also pointing out feeder cell is not by the secretion embryotrophy factor or removes the poisonous factor from substratum, but needs cell-cells contacting to realize.This prompting is worked by integrin receptor, and extracellular matrix components can be regulated the growth and the differentiation of described PGC cell.Making it can not keep undifferentiated state in containing the matrix of extracellular matrix components the PGC colony plating of deriving, is not the problem of simple cell attachment to single cell epimatrix composition thereby point out this.Yet this supports conditioned medium not have the observations (Piedrahita etc., 1990) of positive influence to separating pig ES cell really.

With regard to the vitro differentiation ability, the typical aforementioned simple idiosome of isolating EG colony generation (Martin and Lock, 1983; Piedrahita etc., 1990).Yet they form the limited in one's ability of capsule structure and have only the formation of just finishing utriculi majores when being placed on collagen stroma in suspension.Because vitro differentiation is removed inducing of foetal calf serum and somatomedin, the reduction that might form the cyst ability is owing to the biological chemistry defective in the described matrix, rather than because the developmental defect of described colony.This by the EG colony during mosaic forms in vivo the fact of normal differentiation strengthened.

When analyzing and identify described transgenosis colony, observe the described GFP expression level and the pattern of wide region.A part of having only described colony is that GFP male colony is common, show or described colony derives from a more than cell, and have only one of them is genetically modified, perhaps described genetically modified mix to occur in create a later cell fission period of inlaying colony.Than described inlay colony more in addition people's puzzlement be that the GFP activity is subjected to the fact of the adjusting of cytodifferentiation in some cases.Several colonies are accredited as on by morphology and are in more some part of the colony of differentiation state and express GFP.

A possible explanation this GFP being expressed the adjusting pattern is an observed height location effect when using the CMV promotor.This promotor has the very advantage of high level expression of potential, but the shortcoming that had a strong impact on by contiguous chromatid is arranged, and this influence obtains proof from only being higher than background to very high intensity by the intensity of GFP in the transgenosis colony.This is by being confirmed with the carrier transformed mouse ES cell that contains SV40 promotor that drives the neo gene and the CMV promotor that drives GFP.Behind the electroporation, cell was placed in the G418 selection condition 8-10 days and checked the colony of survival under fluorescent microscope.On average, GFP male neo resistance colony ratio is in the scope of 30-50%.The described CVM promotor of this strong prompting becomes easy silence when mixing.

Simultaneously keep it not break up morphology and AP expression characteristic again in case determined possible genetic transformation PGC derived cell, just can detect versatility in the body of described cell by chimeric generation.The result clearlys show that some mosaics that come by injected embryonic development mix transgenosis in its karyomit(e) with the frequency that 35 tires altogether identify three transgenosis mosaics and 1/3 porkling.Though once attempted by seeking and visiting transgenosis and non-transgenic mouse (0: 100 to 100: 0 with the neo probe, at interval 10%) the typical curve comparison of making and the signal that obtains from the chimeric genomic dna engram analysis of described transgenosis is done sxemiquantitative, the result is then according to intensity of probe, and washing has with hybridization conditions checks the different of limit from 10% to 30%.In any case, find out obviously that from the result of described southern blotting technique analysis and from the detection that GFP a transgenosis tire expresses described transgenosis PGCs can participate in the formation of tire in the growth.

Result described herein shows that PGCs can be separated, cultivates and genetic manipulation and do not have and grow losing of potentiality.Remain certainly though plant system's transmission, preliminary sign shows that described cell can enter sexual gland once more and can show kind of potentiality that are transmission.Therefore, the exploitation of this technology has represented the possibility of carrying out the homologous recombination method in the pig species.

Embodiment 5

The disappearance and the integration of Cre-mediation

The transgenic animal that produce by the injection of conventional pronucleus run into insert inactivation and because regulatory region is not enough and (or) the vicinity chromatid influences the potential problems that are not suitable for the adjusting aspect (Klintworth, 1990) that the position effect of described transgenosis behavior causes.Although position effect can improve to a certain extent by collaborative injection MAR (matrix attachment regions) insulant (Mcknight etc., 1992), described insulating method is neither perfect also not being proved to be has general applicability to all locus.

Can be bypassed by use homologous recombination in embryonic stem cell although insert associated problem at random, this technology is time-consuming, and costliness also can only be used (Gordon, 1993 at present in the mouse species; Stice and Strelchenko, 1995).A kind of alternative method is that transgenosis of guiding arrives an intended gene seat with a dna sequence dna mark, and this dna sequence dna will instruct any following transgenosis to same position.The transgenosis that is integrated (under the regulatory region of target gene seat) should have an expression pattern that is similar to native gene.

Mouse whey acidic protein matter (mWAP) is selected for these research, is because its high mRNA (15% total RNA) and high protein (1 mg/ml) level (Henninghausen and Sippel, 1982a in lactication mammary gland; Grabowski etc., 1991) and because it produces potential importance (Houdebine, 1994 in the generation of proteinic galactophore biological reactor of medical purpose in mammary gland; Yom and Bremel, 1993).Transgene expression thing from this type of locus should have the unique tissue and the pattern of development characteristics, and coded protein is gathered in the crops (Houdebine, 1994) with the high level of non-mode of infection in theory from the Ruzhong.

Site specific recombination system such as Cre-loxP and FLP-FRT have been used with the guiding transgenosis and have entered yeast, plant and the genome (Sauer and Henderson, 1989 that comprise the mammalian cell of ES cell; 1990; Albert etc., 1995; Araki etc., 1997).This method also is used in and produces the tissue specificity rejecting in single ES clone, can induce rejecting and polygene seat to modify (Bradley and Liu, 1996).For utilizing the Cre-LoxP method, the loxP recognition site in the predetermined chromosome position need be introduced into the mouse embryo by the homologous recombination of WAP locus and do (ES) cell.The neo-TK box will be allowed the positive-feminine gender selection of the disappearance incident of carrying out gene targeting and Cre mediation.After the marker disappearance that Cre protein causes, a single loxP locus will remain the genetically modified mark that contains loxP as introducing.

A. materials and methods

1. clone the mWAP gene

Filter out one 14 kilobase (kb) genome WAP clone from a lambda particles phage Charon35 storehouse with an exon 3 specific probe, described storehouse is E14gT2a (obtaining from Nobuyo doctor Maeda of that university of North Carolina) preparation from mouse ES cells.The PCR of mouse WAP ^TMPrimer (forward primer (SEQ ID NO:9): 5 ' TTGGTGTTCCGAAAGCTGGCTTCTG3 '; Reverse primer (SEQ ID NO:10): 5 ' GGGTTATCACTGGCACTGGGGGTGTA3 ') with mouse gene group DNA at PCR ^TMUse under the condition, described condition comprises 94 ℃ of 1 round-robin (5 minutes); 35 94 ℃ of round-robin (1 minute), 55 ℃ (30 seconds), 72 ℃ (30 seconds); 1 72 ℃ of round-robin (10 minutes).The product of a 178bp is purifying on 2% sepharose, the spin column purification and as have [ ³²P]-Random PrimedLabeling test kit (Boehringer Mannheim, Indianapolis, template IN) of dCTP.4.5kb Xhol-EcoI fragment that contains exons 1-4 by subclone to BluscriptIIKS (pBS) (Stratagene, La Jolla, CA) in.

2. plasmid

Plasmid (pWPNT) construction and the employed general plasmid of WAP guiding provide in table 14.(CA), it has a lacZ-cre to the AM-1 cell, is used to check the existence in functional loxP site in the floxed construction for Invitrogen, San Diego.(CA) plasmid that will contain the DNA of loxP site flank is incorporated in the electroreception attitude AM-1 cell and plating spends the night for dull and stereotyped last 37 ℃ at LB-Pyocianil-IPTG (10mM) for BioRad, Hercules with the E.coli pulse instrument.(WI) purifying is used the XhoI linearizing to pWPNT for Promega, Madison, and is stored in-20 ℃ of power supply perforation uses with every part 30 microgram with Wizard MaxiPrep post.

Table 14 plasmid composition pBS64 LoxP site pBS185 CMV promotor: cre gene: MT-IpolyApKJ-1 PGK promotor; R:neo gene: PGKpolyApPGKCre PGK promotor: cre gene: MT-IpolyApHSVCre HSV-TK promotor: cre gene: MT-IpolyApOG231 CMV promotor: synthetic intron: cre gene: MT-IpolyApBSNeo PGK promotor: neo gene: PGKpolyAploxpNeo LoxP:PGK promotor: neo gene: PGKpolyA

PBSNeoTK PGK promotor: the neo gene: PGKpolyAHSV-TK opens

Mover: TK gene: HSV-TKpolyA

PBS185, pBGKCre, pSVCre and pOG231 are used as the source that instantaneous Cre expresses.With regard to the pPGKCre plasmid, employed PGK (phosphoglycerokinase) promotor is from the pKJ-1 plasmid.2.7kb XhoI-HindIII fragment is come the pBS185 of self-contained cre gene, and MT-IpolyA by subclone to pCRII (Invitrogen, San Diego, CA) and be named as pCRIICre.Subsequently, be connected to the pKJ-1 of PstI-HindIII digestion from one 2.7 kb NsiI-HindIII fragment of this subclone.Plasmid pBSNeo-TK is digested to remove described neo box, TK encoding sequence and HSV-TKpolyA signal by PstI.A 2.7kb NsiI fragment from pCRIICre is connected to pBSNeo-TK to produce pHSVCre.

From containing the PGK promotor, the 1.9kb EcoRI-HindIII produced in fragments pBSNeo of neo gene and the pKJ-1 of the PGCpolyA of being cloned into pBS.Enter ploxP2 and prepare plasmid ploxPNeo by cloning a SacII-KpnI fragment from pBSNeo (containing described neo gene).

3. cell tissue is cultivated

Embryonic stem cell (ES) clone AB1 cell (from Allen Bradley, Baylor medical college) is used to full-fledged research.Cell is cultured in the ES substratum, wherein contains to have replenished 15% (v/v) foetal calf serum, the improved Eagle substratum of the Dulbecco of 0.1mM β mercaptoethanol and 2mM glutamine (EMEM).Temperature remains on 37 ℃ of constant temperature in the humidifying incubator that 5% carbonic acid gas replenishes.The ES cell is maintained at that (3400 rads are used caesium by ametycin or gamma-radiation ¹³⁷) on the l cell STO cell of deactivation.Reorganization mouse LIF ESGRO (GibCoBRL), or the people LIF (obtaining from the COS-7 cell of transfection) that recombinates is added in the substratum with about 1,000 units per ml.

Went down to posterity by installing in 100 millimeters sterile petri dish in the every 2-3 of ES cell days with 1: 4 to 1: 10 minute.Wash twice with 3 milliliters of PBS, handle with 37 ℃ in 0.04% trypsinase then and handled in 5 minutes.Add 3 milliliters of ES substratum in the cell of trypsin treatment and disperse described cell to separate any agglomerate.Cell is precipitated and be resuspended in 10 milliliters of ES substratum and equalization is assigned to and contains in the STO flat board.

4.ES the electroporation of cell

A. carry out homologous recombination with WAP guiding plasmid

0.8 the cell (1 * 10 that milliliter is a ⁷) mix with 30 microgram linearizing pWPNT, at 300 volts/250 microfarad electroporations, and plating is in fresh ES substratum, 0.5-2 * 10 ⁶/ flat board.After 24 hours, add contain G418 (200 mcg/ml Geneticins, Gibco BRL, Grand Island, ES substratum NY) is also changed during 10 days selection if needed.Behind the electroporation the 10th day chosen each colony and transferred to asepticly, contains 24 orifice plates of the gelatin bag quilt of ES/G418 substratum.Reaching when being paved with, described cell by tryptic digestion and packing for results genomic dna and-70 ℃ of storages in containing the ES substratum of 10%DMSO.

B. the plasmid with coding Cre carries out the disappearance that Cre mediates

The clone F2C that is directed to is with 30 microgram cre plasmids such as the above-mentioned electroporation that carries out.Because select often observed " bystander effect " with 9-(1,3-dihydroxy-2-third oxygen methyl) guanine, behind the electroporation, cell is with per 100 millimeters flat boards 5 * 10 ⁵Inoculation.At the 3rd day, add 2 μ M 9-(1,3-dihydroxy-2-third oxygen methyl) guanine and select 3 days, use ES substratum feeder cell up to 12-14 days thereafter.

C. with containing the insertion that Cre and loxP plasmid carry out the Cre mediation

Disappearance clone D25 wherein contains the loxP site in a WAP allelotrope, be used to guiding research.With per 0.8 milliliter 1 * 10 ⁶The ES cell 300 volts/250 microfarads carry out electroporation and in new ES substratum with per 60 millimeters flat boards 1 * 10 ⁶Plating.After 24 hours, add the ES substratum contain G418 (200 mcg/ml) and during selecting in 10 days, change during when needs.

5. detect recombination event and dna sequencing

From the isolating genomic dna of ES cell clone be used to the Southern engram analysis and (or) PCR ^TMDiagnosis.With regard to the Southern engram analysis, EcoRI-, the DNA of NsiI-or SphI-digestion be transferred on the N-Hybond film (Amersham, Arlington Heights, IL).With Random Primed Labling test kit (Boerhringer Mannheim, Indianapolis, IN) and use [ ³²P]-dCTP label probe and last Sephadex G-50 column purification.(IL) after 55 ℃ of hybridization, filter membrane was washed 2 * 20 minutes and was washed one time 15 minutes with 1 * SSPE/0.1% (w/v) SDS at 42 ℃ with 2 * SSPE/0.1% (w/v) SDS room temperature for Amersham, Arlington Heights in RapidHyb solution.The filter membrane that is labeled and Kodak's X-Omat AR film are put together and were kept 1-3 days at-70 ℃.

Contain the loxP plasmid and also use the PRISM that contains the AmpliTaq archaeal dna polymerase with Qiagen Plasmid test kit purifying ^TMReady Reaction DyeDeoxy ^TMTerminator Sequencing test kit uses M13 primer order-checking forward or backwards.PCR ^TMComprise that being initially at 96 ℃ of sex change continues 2 minutes 25 96 ℃ of round-robin (30 seconds) subsequently, 50 ℃ (15 seconds) and 60 ℃ (4 minutes).PCR ^TM(Princeton Separations Inc.) and on the 373A automated DNA sequenator of Applied Biosystems Inc. handles Centri-Sep post in the reaction.

The guiding of B.mWAP locus

Displaced type guiding construction pWPNT forms (Fig. 1) by the loxP-Neo-TK-loxP box in the WAP exon 4 of position between termination codon and polyadenylic acid signal.The homology total amount is the 5 ' flanking sequence of 3.0kb and the 3 ' flanking sequence of 1.5kb.Measure the positive by the Southern engram analysis from 199 cloned genes group DNA and be directed to the clone.The size of expection band provides in Fig. 2.Exon 3 is sought and visited EcoRI, and the endogenous band of DNA of NsiI and SphI digestion is respectively 3.9kb, 6.1kb and 7.1kb.The allelotrope that is directed to should have 5.4kb respectively, and 10, the corresponding band of 1kb and 3.9kb.In 16 clones, there are 6 with 5.4kb guiding allelotrope correct guidance from the Southern engram analysis of the DNA of G418 resistance clone with EcoRI digestion.Being directed to clone F2C is proved and has three enzymes that separate.

WAP locus guiding result of study is summarized in table 15.In 199 analyzed clones, 51 are directed to the neo-TK box.Described guiding frequency is determined to be 6.4 * 10 ^-5, and the ratio of homologous recombination and random integration is 1: 4.Different and slightly different in addition in the guiding frequency according to the fate of results.Because the colony that is directed to has the neo gene of single copy, they are slower than the colony growth that the insertion of Duoing is arranged under selective pressure.Therefore, at later time results colony these clones that are directed to are grown if having time.At the 8th day, the colony of picking is positive accounted for 19.6% of described sample, accounts for 27.6% the 10th day clone, and accounts for 26.3% at the 12nd day.

Table 15

Sum up Recomb. #cells Plasmid Amount #cells G418 in the reorganization of WAP locus ^rGanc ^rRecomb/ type treated of DNA plated colonies colonies Analyzed ^* Homologou 1 * 10 ⁷ PWPNT 5nM 2 * 10 ⁶2498-51/199s Recomb.

1×10 ⁷???pBS185??????30μg??????5×10 ⁵????????-?????????1235?????44/50

2.5×10 ⁵??????-?????????1002???????+

1×10 ⁷???pPGKCre?????30μg??????5×10 ⁵????????-?????????1068?????10/10Cre-Mediated????????????????????????????????????2.5×10 ⁵??????-?????????704????????+Deletion

1×10 ⁷???pHSVCre?????30μg??????5×10 ⁵????????-?????????1023?????10/10

2.5×10 ⁵??????-?????????735????????+

1×10 ⁷???No?DNA?????????-???????5×10 ⁵????????-?????????140??????0/10

-???????2.5×10 ⁵??????-?????????135????????+

1×10 ⁶???ploxPNeo+????1μg??????1×10 ⁶????????19??????????-??????11/48 ^

pOG231??????30μg

1×10 ⁶???ploxPNeo+????1μg??????1×10 ⁶????????25??????????-??????5/24 ^

pOG231??????20μgCre-Mediated???1×10 ⁶???ploxPNeo+????1μg??????1×10 ⁶????????21??????????-??????3/24 ^Insertion

pOG231??????10μg

1×10 ⁶????ploxPNeo+????1μg??????1×10 ⁶????????27??????????-??????1/24 ^

pOG231???????5μg

1×10 ⁶????ploPNeo??????1μg??????1×10 ⁶????????33??????????-??????0/10 ^

All electroporations carry out in the ES substratum 200 microfarads and 300 volts.G418 ^r, the G418-resistance; GanC ^r, the Ganc resistance.

^*Positive colony number and PCR ^TM(or) ratio of the colony number of Southern trace determination and analysis.

+ do not determine

++ the each processing uses 1 * 10 ⁶The summation of 3 electroporations of cell.

The disappearance of C.Cre mediation

Use PGK, the effect (table 15) of HSV-TK and the described cre genetic testing of CMV promoters driven promoters driven cre gene.Plating 2.5 * 10 ⁴Cell, 9-(1,3-dihydroxy-2-third oxygen methyl) guanine resistance is handled and the contrast ratio is 8.8: 1 (pBS185); 7.6: 1 (pPGKCre); 7.3: 1 (pHSVCre).With 5 * 10 ⁴The plating cell is handled and the contrast ratio is 7.4: 1 (pBS185); 5.2: 1 (pPGKCre); 5.4: 1 (pHSVCre).Because the relative quantity of 9-(1,3-dihydroxy-2-third oxygen methyl) the guanine resistance colony between twice cell inoculation is approximately identical (2.5 * 10 ⁴To 5 * 10 ⁴), the influence of " bystander effect " can be by reducing with low extent of dilution plating cell.

With regard to PCR ^TMConfirm the disappearance incident, primer (270bp) allelotrope that endogenous (166bp) but also amplification are modified that is designed to not only to increase.The 0.7kb BamHI-PstI WAP fragment of the downstream sequence of 3 ' terminal and about 650 bases that contain exon 4 is cloned into Bluscript IISK and with Sequense Version 2.0 sequencing kits (the U.S.Biochemical Corporation that checks order, Cleveland is OH) to find PCR ^TMPrimer (forward primer (SEQ ID NO:11:5 '-AGCGACCAGCCCAAGTGTATACAG-3 '; Reverse primer (SEQ ID NO:12): 5 '-GCCTGCTTTGTCGTTCCTTCAG-3 '; ) they are positioned at the flank in loxP site.PCR ^TMReaction conditions is 94 ℃ of 1 round-robin (2 minutes); 33 94 ℃ of round-robin (30 seconds), 54 ℃ (30 seconds), 72 ℃ (30 seconds); 1 72 ℃ of round-robin (10 minutes).

50 clones' PCR ^TMAnalyze and show that 44 have allelotrope (88%) endogenous and that modify.PCR ^TMShow that with the Southern engram analysis great majority clone contains the correct PCR of loxP site male ^TMProduct.Described 166bp and 270bp band also are WAP exon 4 sequence male.Described 270bp PCR ^TMProduct is checked order and has been found wild-type loxP site.With SphI the Southern analysis that deletion clone carries out is shown that 3.9kb is directed to losing of band; Adorned locus is big or small identical with 7.1kb endogenous band.

The insertion of D.Cre mediation

1 microgram loxPNeo and 30 microgram pOG231 collaborative electroporation in the ES of loxP mark cell causes each average 19 G418 of electroporation in 3 electroporations ^R(by contrast, the ploxPNeo control group has 11 G418 to colony ^RColony) (table 15).Although the plasmid of coding Cre has nothing in common with each other G418 ^RThe sum of colony (comprise at random with site-specific nature intasome) is constant substantially in entire area: use 5 micrograms to obtain average 27 G418 ^RColony; 10 micrograms obtain 21 G418 ^RColony; 20 micrograms obtain 25 G418 ^RColony; And the background contrast is 12 colonies.The ability that other construction of expressing Cre mediates the insertion incident is determined.PCR ^TMAnalysis has pBS185, the G418 of pPGKCre and pHSVCre plasmid ^RColony shows that not having colony to contain site-specific nature inserts fragment.And, G418 ^RThe number of colony is irrelevant with the collaborative electroporation of coding Cre plasmid.

PloxPNeo guiding plasmid is integrated loxP flank PGK promotor, neo gene and the PGK polyadenylation signal box of the exon 4 that will cause being arranged in mouse WAP gene in WAP locus site specificity.Produce a PCR who is combined with about 750bp of WAP exon 4 and neo primer with the ploxPNeo guiding ^TM Band.WAP exon 4 specificity primers (SEQ ID NO:13:5 '-AGCGACCAGCCCAAGTGTATACAG-3 ') and Xin Meisu specificity primer (SEQ IDNO:14:5 '-TGACCGCTTCCTCGTGCTTTAC-3 ') are to being used to the PCR in the genomic dna diagnostics ^TMDiagnosis, described diagnosis is carried out under the following conditions: 94 ℃ of 1 round-robin (2 minutes); 94 ℃ of 33 round-robin (30 seconds), 55 ℃ (30 seconds), 72 ℃ (30 seconds); 72 ℃ of 1 round-robin (10 minutes).

At 48 G418 that analyze from the collaborative electroporation of ploxPNeo-pOG231 ^RIn the colony, 11 is the site-specific nature integron.Seven representative samples are male, have the correct 750bp band from the ploxPNeo plasmid integration.Inserting the quantity that frequency is found directly with the pOG231 of electroporation changes.When representing with per-cent, the site-specific nature integrating frequency be 30 microgram pOG231 22.9%, 20 microgram 20.8%, 10 microgram 12.5%, 5 microgram 4.2%.

The WAP locus that ploxPNeo is integrated into exon 4 produces the SphI band of the 4.8kb of an expection, and endogenous WAP locus is 7.1kb.Because the neo gene of ploxPNeo and pWPNT is cloned with different orientation, produce a 3.9kb band with pWPNT homologous recombination guiding WAP locus.In contrast, the clone that is directed to has the allelotrope of a 3.9kb, and disappearance clone has a 7.1kb allelotrope.Except a clone (may be the compact land of described insertion incident), whole PCR ^TMPositive colony obtains certainly by southern blotting technique.

Result discussed above has described the Cre-loxP system has been used to fix a point to insert the operability of a dna fragmentation to the predetermined chromosome position.Though selected locus reflection inventor can be used as the interest of the transgenic animal aspect of mammary gland biologically device to generation, it all is available that entire method is tackled any goal gene seat.

So far, this is the report of first guiding WAP locus and also is the integrating remark of first Cre mediation in predetermined loxP locus ES cell.This work confirms that this incident takes place with the level near homologous recombination really.Therefore, inject the transgenic animal that produce by the blastocyst of this ES clone a loxP target for the transgenosis insertion of Cre mediation can be arranged.Because position, described loxP site is between WAP terminator codon and polyadenylation signal, the transgenosis that contains internal ribosome entry site (IRES) can be used to produce WAP-transgenosis bicistronic mRNA courier (Pelletier and Sonenberg, 1988).This method can be avoided being expressed and any unpredictalbe problem of causing by whole elimination WAP; In addition, be equipped with the genetically modified transgenic animal of single copy in identical bits and should produce consistent expression pattern.

Relate to the great majority application of Cre-losP system in the Mammals environment and be fit to utilize its efficient when disappearance loxP flanking DNA sequence.Before get the result and show that this incident can take place at ES cell high frequency from the disappearance institute that Cre mediates.In inventor's research, about 2.2% the electroporation that is directed to cell that carries out with pBS185 is with every dull and stereotyped 5 * 10 ⁵Stood the disappearance of described neo-TK box during inoculation.In similarly studying, the J of IgH locus in the ES cell _H-E _μLead with the neo-TK box of loxP-flank in the district.The carrier pIC-Cre of coding Cre and pMC-Cre contain enhanced translation and nuclear localization signal respectively, and they produce with this inoculum density and are equivalent to 2.0% and 4.0% value (Gu etc., 1993).Select with FIAU, Abuin and Bradley (1996) find 16% the disappearance that cell has experienced loxP flank selection box that is directed to, and this phenomenon has proposed query to using selective pressure in the necessity of disappearance incident.In view of the above, use recombinant adenovirus to express Cre and cause almost 100% the culturing cell (Kanegae etc., 1995) that has lacked the loxP-flanking sequence that has.

Yet, the more important thing is that Cre-loxP and FLP-FRT system shown and cause that the DNA site-specific nature is integrated into Mammals karyomit(e), carry out to be lower than the frequency that described disappearance incident found although be.The reorganization that O ' Gorman is presented at the mediation of FLP in the monkey-kidney cells produces than the high about 2 times level of random integration (O ' Gorman etc., 1991).Baubonis and Sauer (1993) use the instantaneous source of Cre protein as recombinase in the human osteosarcoma cell line of loxP-mark.Use the promotor trap system, they find a kind of " position effect ", demonstrate 50 times difference because contain the loxP site clone of random integration on the guiding frequency.The existing report of integration in the ES cell in use wild-type loxP site is site-specific nature (Araki etc., 1997) although the integron of less than 0.5% is arranged.It is 23% (48 G418 of random integration that the site-specific nature that has been presented at described WAP locus from inventor's research is integrated ^RThere are 11 in the colony).Since described WAP locus by homologous recombination with 1: 4 HR: (homologous recombination: non-homogeneous reorganization) be directed to, the incident of described Cre mediation takes place with the level approximately identical with homologous recombination the NHR ratio.

Though insertion and homologous recombination frequency in described WAP locus Cre mediation in this research are similar with respect to inserting at random, site-specific nature inserts some additional advantage.For the time based on the insertion design construction thing of Cre, the loxP site that unique needed homology is 34bp (with thousands of base ratios of conventional gene targeting than).In addition, described loxP site mutation can make inserts frequency above the insertion frequency (Albert etc., 1995) that is obtained with wild-type loxP site, shows the frequency that might further increase the insertion incident thus.Yet after described integration incident, the complementation sudden change in two loxP sites will cause wild-type and dual sudden change loxP site.This dual sudden change loxP site will not be subjected to the constraint of Cre, thereby can not participate in increasing whole excision reaction of inserting frequency.As recent demonstration, the insertion that the loxP site Cre that uses this strategy can make at random site in the ES cell mediates increases by 30 times (Araki etc., 1997).

And as shown in Table 15, the scale effect recombination fraction of plasmid of transgenosis and coding Cre is indicated as the optimum proportion that reaches that reaches between the maximum transgenosis of inserting frequency and need Cre level and loxP mark.To inserting an index of incident importance, unique plasmid that causes detecting the expression Cre of integration incident is pOG231 as the Cre level.When comparatively high amts pOG231, the integration of seen higher level infers it is because the increase of pOG231 concentration in each cell.The plasmid that relatively is used for the coding Cre of neo-TK box disappearance shows that 9-(1,3-dihydroxy-2-third oxygen methyl) the guanine resistance colony number of pOG231 is higher 4 times than pBS185.Described Cre gene be placed on known in the ES cell integrating frequency to determine whether can to cause like this to increase under the activated different promoters.Therefore, there are several potential methods to increase and insert frequency to surpassing the level that this paper reported and this system (or modification mode of this system) being become producing the very useful system of transgenic animal.

At present, there is not other alternative method of directly on embryo, using, because conventional gene targeting was not only very invalid but also follow the variation (Brinster etc., 1989) of foreign DNA in embryo.Previous existing report Cre protein plays disappearance loxP-flanking sequence (Lakso etc., 1996) in a cell stage mouse zygote.In mouse, the efficient that produces transgenic animal by the pronucleus injection is 5-20% (the young mouse sum of the transgenosis children mouse/birth of birth).If be similar to ratio in the ES cell of cultivation in the ratio of inserting with random integration in the embryo of the transgenosis of loxP mark and the collaborative injection of Cre enzyme and site-specific nature, the insertion that the transgenosis children mouse between the 1-5% that is born will have a Cre to mediate at described marker gene seat.This will cause a kind of form by the modification of pronucleus injection guiding.At present, the inventor just producing have be labeled the WAP locus mouse to study this effect.

By introducing termination signal to described WAP encoding sequence, this system also is suitable for macrofauna.This method produces bicistronic mRNA information, from IRES output target protein matter, but does not produce described WAP protein.This is useful, because the injury of mammary gland in mouse WAP protein and the transgenic pig is related.

Embodiment 6

The separation of pig apolipoprotein E gene and sign

Apo E (apo-E) is a kind of composition of all kinds of plasma lipoproteins in the mammalian body, and it has several main effects, comprises transportation and the metabolism (Mahley, 1988) as phosphatide and triglyceride level of cholesterol and fat.In human body, comprise 299 amino acid whose mature polypeptides (Rall etc., 1982) and be synthesized to containing the preapoprotein-E of one 18 amino acid signal peptide, this signal peptide is when translation cut (Zannis etc., 1984).Apo-E is synthesized in many places, comprises liver, brain, and spleen and kidney, liver is the maximum producer (Mahley, 1988).Synthetic peripheral cells such as the scavenger cell (summary of Getz etc., 1988) of also occurring in.Its cDNA and genome nucleotide sequence be all known to be limited to several species, comprises people (Das etc., 1985; Paik etc.; 1985), mouse (Rajashisth etc., 1985), rat (Fukazawa etc., 1986) and baboon (Hixson etc., 1988).

And lipoprotein receptor such as apo-B as if the mechanism of action of apo-E in cholesterol and metabolism of fat is complicated and, E (LDL) acceptor interaction is about (Mahley and Inneratity1983).The mankind, the zone that the 140th to 160 amino acid vicinity is rich in arginine and Methionin is receptor binding domain (Inneratity etc., 1983; Weisgraber etc., 1983).Single amino acid in this zone is replaced the inheritable variation that causes the apo-E locus.Some abiogenous variation presents defective type and relevant with the cardiovascular disorder of III type hyperlipoproteinemia and acceleration (Weisgraber etc., 1982) of receptors bind aspect.In the 112nd amino acids is respectively that halfcystine and arginic apo-E3 and apo-E4 isotype have normal receptor binding capacity (Weisgraber etc., 1982).

The effect of apo-E in atherosclerosis takes place is complicated and is difficult to study the mankind (Getz etc., 1988).Because cardiovascular and gastrointestinal physiology and the people of omnivore are similar, pig extremely is suitable for studying cardiovascular disorder, and (Hodson 1985; Armstrong and Heistad 1990).In fact, there be (referring to Swindle 1992) in the cardiovascular disorder pig model of all respects.The pig model that shows the atherosclerosis situation is very attractive to the scientist of the human diseases of studying this complexity.The separation of pig apo-E gene and sign make this method walk close to real stepping and go a step further.

A. the clone of pig Apo-E gene and order-checking

Be used for from mouse apo-E cDNA's ³²The 700bp SacI/BglII fragment (Piedrahita etc. of P mark, 1992) fragment (8-22kb) that partly digests by Sau3A1 of screening cloned the BamHI site that enters λ carrier EMBL3 Sp6/T7 (Clontech LaboratoriesInc.Palo Alto, CA) and the pig genomic library (Sambrook etc. 1989) of structure.Isolate a positive phage clones and obtain a 10.7kb DNA and insert fragment with EcoRI/SalI digestion back.Then, with the method for having set up (Sambrook etc., 1989) this 10.7kb dna fragmentation is cloned among phagemid Bluscript (pBS) M13.

Insert the position of apo-E gene in the fragment and the subclone of being convenient to check order for locating this 10.7kb, make this clone's part restriction endonuclease map.Contain complete pig apo-E gene and be positioned at 4.2kb XhoI/XhoI fragment that described 10.7kb inserts fragment 3 ' end by subclone in pBS.The subclone that produces overlapping is for the usefulness from the XhoI/XhoI cloning and sequencing.With two the chains order-checkings of the general forward of M13-20 and 17 aggressiveness reverse primers to described subclone.Use Dideoxy ^TMDyeTerminator/Sequenase ^TMTest kit (Applied Biosystems Division, Perkin-Elmer Cetus, Emeryville, CA USA) uses Applied Biosystems377 dna sequencing instrument to check order.MacVector ^TMAnd Assemblyline ^TMProgram is used to comparison and contrast sequence data.

Screen the pig genomic library and identify the single positive phage clone with mouse apo-E-specific molecular probe.Separate described clone, and insert fragment by discharging 10.7kb with EcoRI and SalI digestion.The described insertion fragment of restriction endonuclease mapping and Southern analysis revealed contains complete pig apo-E gene at the segmental 3 ' end of its 4.2kbXhol/Xhol.Determine described nucleotide sequence (SEQ ID NO:5) and pig apo-E gene deduced amino acid (SEQ ID NO:6) then.

The similar of pig apo-E gene is in people (Das etc., 1985; Paik etc., 1985), mouse (Rajavashisth etc., 1985), the structure of rat (Fukazawa etc., 1986) and baboon (Hixson etc., 1988), it also is made up of four exons that separated by three introns.The size of exons 1 to 4 is respectively 26bp, 66bp, 190bp and 843bp.The size of three introns is respectively 804bp, 744bp and 374bp.What is interesting is that the size of exon 2 (66bp) in these species that checked order is identical.The size of other exon is inequality at these species.

4, determined its complete sequence of 266bp Xhol/Xhol fragment (SEQ ID NO:5; GenBank number of registration 470240).Except 3057bp pig apo-E gene, it contains the 3 ' flanking region of 5 ' and the 378bp of 831bp.The exon border is by (Brzozowska etc. 1993a) are relatively identified with the previous cDNA sequence of measuring with the pig nucleotide sequence.The exact position at described intragenic three introns has been determined in the evaluation of intron splice site consensus sequence 5 ' GT and 3 ' AG.

Described gene comprises the 5 ' non-translational region of 49bp, comprising exons 1 and part exon 2, connect surplus encoding sequence crossing over exon 2 down to exon 4,18 amino acid whose signal peptides that this encoding sequence coding is inferred, 299 amino acid whose mature proteins and the 125bp end of 3 ' non-translated sequence is arranged.First exon and intron appear in the 5 ' non-translational region (position is respectively at G1 and t28).Second intron interrupts the codon glycine in signal peptide aminoacid sequence (G897)-4 position.The 3rd intron interrupts the Threonine codon in the position 60 of mature protein (G1831).The mRNA of 1125bp is by pig apo-E genes encoding.

All (Brzozowska etc. 1993a) relatively the time, find to have 17 Nucleotide differences with the previous cDNA that measures when its aminoacid sequence of exon nucleotide sequence and supposition.Yet, 10 not influences of aminoacid sequence during these Nucleotide are replaced to the described mature protein of supposition.Wherein, be positioned at outside the described coding region 8 at

nucleotide position

1,2,5,8,9,252,253,254, and two positions 2470 and 2893 in exon 4.7 replacements in described mature protein amino acid position 17 for S to P, 142 for N to K, 143 for V arrives L, 148 arrive L for V, 176 for L arrives F, and 233 for D arrives E and 234 for E arrives Q, corresponds respectively to nucleotide position 1701,2455,2456,2470,2471,2728 and 2729.Inventor's sequence is contained in the additional CCC of nucleotide position 2952 beginnings, the 1122bp that it causes 1125bp (1993a) such as mRNA replacement Brzozowska to find.The difference that this paper finds can not obtain because sequence data is two chains of cloning from overlapping because the order-checking illusion causes.

Described mature protein has L-glutamic acid as its NH ₂Terminal residue, its codon start from the 1653rd of described nucleotide sequence.The interior standard AATAAA of 3 ' non-translational region (Benoist etc., 1980) that appears at most of eukaryotic genes is positioned at 21 Nucleotide places, exon 4 terminal upstreams.

Use MacVector ^TMProgram is with aminoacid sequence and people (Das etc., 1985 of the pig primary translation product of supposition; Paik etc. 1985), baboon (Hixson etc., 1988), monkey (Marotti etc., 1989), and milk cow (Brzozowska etc., 1993b), mouse (Rajavashisth etc., 1985), rabbit (Hao etc., 1987) and rat (Fukazawa etc., 1986) arranges contrast.Arrange contrast for carrying out maximum, the amino acid place that need lack in some species introduces the gap.These gaps mainly occur in mouse, in terminal preceding 37 amino acid of the NH2 of rabbit and rat apo-E.In addition, the rat sequence has been lacked 6 amino acid at its COOH end.

The whole amino acid similarity of pig apo-E and each other species is as follows: the people 70.3%, baboon 72.3%, monkey 70.3%, milk cow 72.2%, rabbit 68%, mouse 65% and rat 60%.The aminoacid sequence of above-mentioned each species is arranged contrast mutually.The amino acid conservative property of higher degree obviously is in those closely-related species, and the people has 93% amino acid similarity, and 91% amino acid similarity is arranged between mouse and the rat between baboon and the monkey.

Pig is inconsistent at the aminoacid sequence of 32 positions and some or all other species.And, be non-conservative near 50% in these aminoacid replacement.Than the amino acid in its end (20-40 amino acid and 212-299 amino acid) higher similarity is arranged at described proteinic middle part (41-211 amino acid) between species.The similarity scope that exists in 18 amino acid signal peptides between the species is 50% to 70%, and the amino acid similarity scope in ripe proteinic preceding 22 amino acid is 19% to 36%.By contrast, the similarity scope that exists between residue 41-211 is 75% to 90%.

Except the difference of 18 amino acid positions, the corresponding sequence of the cDNA of (1993a) reports such as the exon sequence of this paper report and Brzozowska matches.By contrast, circumstantial evidence prompting at least some difference be attributable to mispronouncing of in (1993a) sequences such as Brzozowska base.With regard to this paper genome sequence, infer that be consistent at 7 locational 6 amino acid (amino acid/11 42,143,148,176,233 and 234) that cause amino acid to be replaced with the corresponding section that is arranged correlated seven species all or almost all.In the 17th amino acids that described mature protein is inferred, it is proline(Pro) that the 7th of genome sequence coding is replaced, and cDNA is a Serine.The conservative degree in this zone is much lower and have only Niu Zaidi 17 for being proline(Pro), is Serine and there are not other species in this position.

And two sudden changes (at amino acid position 142-143,147-148 and 233-234) are uncommon, and most of amino acid replacements are that monamino acid is replaced (Mclean etc., 1984) in the eukaryotic gene.The polymorphism of nature is the difference of unlikely explanation two pig sequences.And (Brzozowska etc. afford food for thought when 1993a) comparing for the additional CCC nucleotide triplet that begins in genomic 2952 positions and the corresponding section of cDNA sequence.It is positioned at 3 ' non-translational region and does not cause translation to read the position of frameing shift.Yet, as if the genome sequence of this paper and (Brzozowska etc., cDNA sequence 1993a) is all summarized with the suitable Mahley of apo-E4 isotype of Human genome, 1988 because both all have 111 of mature proteins and 157 s' arginine.On the other hand, the comparison between human apo-E and other species may be inappropriate, because 61 arginine (only finding in the mankind) play a crucial role in decision E4 isotype (Dong etc., 1994).

The length of the exons 1 in pig be approximately the people (Das, etc., 1985; Paik etc., 19985), baboon (Hixson etc., 1998), monkey (Marotti etc., 1989), 60% of mouse (Rajavashisth etc., 1985) and rat (Fukazawa etc., 1986).Obviously, this is not crucial when determining described protein length, because it is not translated.Comparatively speaking, the length conservative property of exon 2 may be the common needs of coding most of signal peptides institute between the species.This may be with handed down from one's ancestors relevant, mentions indirectly as (1985) such as Paik, and they found once that the length of other member (being apo-AI and apo-CIII) exon 2 of this gene family was much at one in apo-E.The amino acid of preceding 18 suppositions of pig apo-E meets the apo-E signal peptide of the mankind (Zannis etc., 1984) and other mammalian species (Yang etc., 1991).Hydrophobic amino acid is rich in this zone, and this is the feature (Verner and Schatz, 1988) of precursor peptide, and conservative relatively between planting.There is 73% consistence between people and the pig, and has 67% consistence between people and the rat.The ripe apo-E length of pig (299 amino acid) and people's consistent (Rall etc., 1982), and than ox (Brozowska etc. 1993b) grow an amino acid.

The regulatory element that people apo-E gene is existed has carried out extensive analysis (Paik etc., 1988; Smith etc., 1988; Chang etc., 1990).The Computer Analysis of the 5 ' adjacent domain of pig apo-E show with people apo-E in find different, the adjusting of this genetic expression is a complex process (Smith etc., 1988) beyond doubt.These investigators find that being not less than 15 zones is protected by the DNA footprint.Smith etc. (1988) infer that this complicated adjusting form provides the expression of apo-E in histological types (depending on cell inner cholesterol concentration) and other nutrition and the hormone factor.Simonet etc. (1991) find that the downstream influences the expression of apo-E in the human liver as far as the regulatory element of 14kb.

The TATA box of finding in most of promoter in eukaryote occurs in and human identical position (Paik etc., 1985).Yet the transcription initiation site of inferring among the pig apo-E is the 20bp place, downstream in the mankind.This may be the short reason of exons 1 of pig.Also see (Smith etc., 1988) in the rat apo-E gene with the similar sequence motif of in the mankind, finding of whole apo-E elements.The Computer Analysis of described pig gene shows some sequence and the corresponding sequence found in the 5 ' zone match (Smith etc., 1988) in people apo-E gene.

Outside element more common in most of eukaryotic genes such as TATA and GC box, the adjusting sequence of other supposition also navigates to 5 ' adjacent end of pig apo-E gene.Apo-E is high expression level (Mahley etc., 1988) in liver, therefore, finds that TCATACTC sequence and liver specificity enhanser protein C/EBP_cs2 (Costa etc., 1988) combination is not unexpected.Described apo-E gene is also expressed and involved in brain is a candidate Alzheimer diseases predisposing gene (Pericak-Vance and Haines 1995).Regulate consensus sequence (TCTGTCTC) with the enhanser of two kinds of protein bound in brain, finding especially and be considered to start from-435 of pig apo-E.Because the complicacy that apo-E regulates, adjusting sequence in addition may be present in the pig gene because to the upstream regulatory sequence of pig seek and visit at present and not exhaustive, and the possible sequence of being found remains to be studied by the DNA footprint and confirms.

Limited protease digestion to people apo-E produces the peptide fragment that two classes have remarkable different physics and chemical property.One class extends to the 191st from first amino acids and has represented the N-terminal functional zone, and another kind of be the C-terminal functional zone from residue 216 to 299.The former and latter's peptide contain reporter gene and fat combined function district (by Mahley summary, 1988) respectively.The apo-E sequence of seven above-mentioned species and the arrangement of pig contrast show an amino acid conservative mode with distinct contrast between these two zones.Is very conservative from the N-terminal functional zone of residue 20 to 211 having the conforming species of 75% to 90% amino acid.C-terminal functional zone conservative property is much lower.In addition, the 260th to 280 amino acid in the C-terminal functional zone is very conservative between species; This may be relevant with function.Sequence between 140 to 160 is crucial (Lalazar etc., 1988) to receptors bind.Pig is consistent with people's sequence except that two non-key positions in this zone.Clay etc. (1995) find that 141 to 149 amino acid region has two halfcystines, and halfcystine is influential to interleukin II dependent form T lymphopoiesis.Measure these influences and whether be applicable to also that pig apoE is significant.

B. determine (CG) ₁₃The polymorphism of little satellite locus

A simple sequence repeats (microsatellite marker) and is detected in the introne 3 of pig apo-E gene.Use MacVector ^TM5.0 programdesign and iteron flanking sequence complementary locus-specific primer.Employed primer is respectively forward primer (SEQ ID NO:15:5 '-AGCTGCTCAGCACCAAGGTCAC-3 ') and reverse primer (SEQ ID NO:16:5 '-CTGAGGGTCCAGACCACACGG-3 ').Belong to four pig varieties (Yorkshire, Landrace, Hampshire and Duroc from 40 affinity-less relations; 10 of every kinds; Larry doctor Shook acquisition from University of Minnesota) animal gene group DNA passes through PCR ^TMAmplification is to measure interracial polymorphism degree.

PCR ^TMCondition is: the genomic dna of amplification 50ng in 50 microlitre volumes wherein contains each primer of 50pmol, 3mM magnesium chloride, every kind of dNTPs of 200 μ M and at Standard PC R ^TMThe Taq polysaccharase of 2.5 units in the damping fluid/0.5 microlitre (Promega, Madison WI, USA).Cycling condition is: 94 ℃ continue 2 minutes, and 35 round-robin continue 30 seconds for 94 ℃ subsequently, and 61 ℃ continue to continue 30 seconds in 30 seconds and 72 ℃.The product of one 194 Nucleotide is expected and this primer pairing.Described forward primer be before PCR with [γ- ³²P]-ATP mark 5 ' end.When pcr amplification stops, add 0.75 volume gel and upload damping fluid (95% methane amide, 0.05% dimethylbenzene green grass or young crops, 0.05% tetrabromophenol sulfonphthalein, 0.5M EDTA) in each reaction, then it is continued 5 minutes at 90 ℃.On 6% denaturing polyacrylamide gel on the standard sequencing gel device, 40 watts, 45 ℃ were carried out electrophoresis 2.5 hours.After the electrophoresis, dry glue is 2 hours in 80 ℃ of vacuum, and the radioautograph exposure.

Simple sequence repeats or (CG) ₁₃Little satellite is detected in the pig apo-E gene intron 3 that starts from the 1856th of described nucleotide sequence.Be somebody's turn to do (CG) ₁₃Little satellite is medium polymorphism.In detected 40 animals, be that to detect size be 190,193,194 and four allelotrope of 199 bases on the basis with the electrophoretic mobility from four pig varieties.In eight animals, detect 4 allelotrope from the Yorkshire kind.Polymorphism information content value (pic) is 0.58, and described gene frequency is 0.38 (190), 0.13 (193), 0.44 (194) and 0.06 (199).

Three allelotrope is present in eight animals from the Hampshire kind.The pic value is 0.54 and gene frequency is 0.31 (190), 0.50 (194) and 0.19 (199).The pic value of Duroc is 0.51, and gene frequency is 0.50 (190), 0.38 (194) and 0.13 (199).The pic value of Landrace is 0.35, and gene frequency is 0.67 (190), 0.34 (194).All detected allelotrope 190 and 194 is frequent appearance in all four strains, and allelotrope 193 is that the Yorkshire kind is exclusive.

This is to have one (CG) in the introne 3 of pig apo-E gene ₁₃The reported first of microsatellite marker.This little satellite is detected in pig at the species that carry known apo-E genome sequence.Compared by the test product kind with other three, the reason of the higher polymorphism degree seen in the Yorkshire kind it be unclear that.The number of this result and animal subject is irrelevant, because the animal of eight affinity-less relations of each kind is a kind of enough big sample sizes.And this may not be unique to Yorkshire, and may extend into the kind that other is tried in this research.The importance of identifying new microsatellite marker in the genome of domestic animal species can not be omitted.Effort at present is in process, in order that the high-density linkage map of exploitation pig is to seek locus (Rohrer etc., 1994 of the influence phenotype of being paid close attention to; Rettenberger etc., 1995; Robic etc., 1995).

C. measure pig Apo-E chromosome position by FISH

The pig Metaphase Chromosome is that the cytogenetic methods of following standard stimulates the lymphocyte preparation from phytohemagglutinin.In situ hybridization fluorescence (FISH) method is carried out (Pinkel etc. 1986) according to standard method, omits inching (Gallagher etc., 1993) as described previously.From the DNA of the lambda particles phage clone purification that contains complete apo-E gene (insert and carrier) by vitamin H by the nick-translation mark.The pig total genomic dna that is labeled probe and excessive cut-out of 200ng is dissolved in the 10 microlitre hybridization solutions.Under the cover glass of sealing, 37 ℃ of hybridization are proofreaied and correct and are spent the night.After post-hybridization washing, with the probe of the plain mark of the plain detection of biological of FITC link coupled affinity.Then, the FISH prepared product is fixed in the anti-solution that fades that contains counterstain iodate third ingot and Hoechst33258 (every kind of concentration is 500-700ng/ml).Show that with the color film sequential shoot PI that is produced by the Hoechst counterstain redyes the photo in mid-term that adds FITC probe signals and QFH band.The QFH band karyomit(e) that shows probe hybridization is identified (the tame pig stdn caryogram council 1988 according to tame pig GTG-band standard caryogram; Yerle etc., 1991).

The lambda particles phage clone that use contains pig app-E gene carries out FISH and the apparent band of QFH as probe order on pig Metaphase Chromosome smear.Analyzed altogether 30 mid-term smear, and 21 smears have shown strong kinetochore specific hybrid signal on the 6th karyomit(e).Wherein, 38% (8 smears) all shows symmetric hybridization signal (two pairs of macula lutea points) on chromatid and karyomit(e); 14% (3 smears) has three macula lutea points (hybridization signals on 3 right chromatids of homologous chromosomes); 29% (6 smears) shown symmetric signal (two macula lutea points) on chromosomal two chromatids; And 19% (4 smears) has a spot on a chromatid.All the yellow spotting performance is positioned at the 6th karyomit(e) centric region and long-armed 2 with the specific hybrid signal between 1 subzone.

The ratio of brachium is calculated with 20 Metaphase Chromosomes, and 12 (60%) shows that pig apo-E gene is positioned on the 6th karyomit(e) (all the other eight chromosomal data it be unclear that).It is to arrange contrast and be proved (tame pig stdn caryogram the council, 1988) by the 6th karyomit(e) standard idiogram of the QFH banding chromosome of FISH mark and pig and G being shown band that Apo-E is positioned on the 6th karyomit(e).

Embodiment 7

The separation of pig (CNTF) gene characterizes and chromosomal localization

Ciliary neurotrophic factor (CNTF), a kind of survival factors (the Barbin etc. that are considered to the chicken parasympathetic neuron at first, 1984), similar having demonstrated of other member of it and hematopoietic cytokine family kept the ability (Conover etc., 1993) that the mouse embryo is done (ES) cell in cultivation.By also forming six aggressiveness that comprise a gp130/LifR β heterodimer (Desirio etc., 1995) with its acceptor interaction, CNTF helps activating gp130 signal transduction pathway (Ip etc., 1992; Stahl etc., 1993), this approach finally causes described ES cell undifferentiated, the phenotype of propagation (Yoshida etc., 1994).Owing to shown allos CNTF already the separation that the pig embryo derived cell that characterization ES morphocytology is arranged is there were not active effect (Moore and Piedrahita, 1996), the inventor clones pig CNTF, whether order-checking is mapping also, as determining to use homologous protein to the more favourable the first step of inhibition of early stage pig embryonic cell differentiation.

A. the clone of pig CNTF gene and order-checking

Be structured in pig genomic library among the λ EMBL3 SP6/T7 (Clontech) with the 369bp probe screening of the exon 2 of whole exons 1s that contain pig CNTF cDNA and 253bp.Described probe is by the reverse transcriptional PCR (RT-PCR of pig activated scavenger cell mRNA ^TM) produce.5 ' primer (SEQ ID NO:17; 5 '-GGATGGCTTTCGCAGAGCAAACAC-3 ') and 3 ' primer (SEQ ID NO:18; 5 '-GCTGGTAGGCAAAGGCAGAAACTTG-3 ') conserved regions that is tested and appraised rat and people CNTF is synthesized.Use this probe, identify seven positive plaques and take turns with third round screening and determined a single positive plaque by second.

This positive plaque is cultured in the suspension and uses standard method DNA isolation (Sambrook etc., 1989), and subsequently with several restriction enzyme digestion.Behind the Southern trace, identify one and contain the 6kb ApaI fragment of complete CNTF gene and be cloned among the pBluescript.From this clone, forms four less, the secondary clone of overlapping is with the complete cycle sequencing that carries out pig CNTF of facility mutually.Described secondary clone, its name with and the trans-regional HindIII of comprising 700bp (containing 5 ' non-translational region (UTR) and part exons 1); HindIII 3kb (end to 3 that contains exons 1 ' UTR); XbaI 1.7kb (contains the introne 1 that is lower than 140bp, exon 2 and 3 ' UTR; With DraI 1kb (containing 50bp exon 2 and 3 ' UTR approximately at least).The dyestuff that whole four secondary clones are carried out two chains stops that repugnancy checks order once more with the correct sequence of unquestionable affirmation between thing cycle sequencing and two chains.

Check order with M13-20 and reverse primer and other primer of designing for the unreachable zone of M13 primer.Use the DNA of QIAGEN Plasmid test kit preparation for order-checking usefulness.The PCR that is comprised ^TMReact as follows: 1 microgram ds plasmid, 8 microlitres and AmpliTaq  FS (AppliedBiosystems, Foster City, CA) reaction mixture and 3.2pmol primer, cumulative volume are 20 microlitres, and sex change in initial 2 minutes, 96 ℃ continue 30 seconds then, and 50 ℃ continued 15 seconds and 60 ℃ lasting 4 minutes, and circulated 25 times.Use Centri-Sep ^TMPost removes excessive nucleosides acid and direct dyes and stops thing, and analyzes described PCR with ABI PRISM377 automated DNA sequenator ^TMReaction.Use MacVector ^TMAnd AssemblyLine ^TMEditor also arranges contrast.

Pig CNTF gene comprises two exons, is separated by the 1258bp intron.Exons 1 is the 114bp length and 38 amino acid of encoding, and exon 2 is the 486bp and all the other 162 amino acid of described CNTF protein of encoding.Intron/exon border is by relatively determining with people CNTF and determining by the position of 5 '-GU and 3 '-AT splice site.The analysis revealed TATA box of promoter region in-54bp position and CAAT box at-120bp.Though both are inconsistent a bit with the consensus sequence of these elements of report, they with people CNTF promotor in identify consistent and be in identical relative position (Negro etc., 1991).Described TATA box is positioned at the just position identical with people CNTF, and 1bp has been moved in the demonstration of described CAAT box downstream.PolyA adenosine acidifying site (AAUAAA) is positioned at 368bp place, terminator codon downstream (GenBank number of registration U57644).

Pig CNTF amino acid and cDNA nucleotide sequence and rabbit (Lin etc., 1989), rat (Stockli etc., 1989), the sequence of people (Negro etc., 1991) and mouse (GenBank number of registration U05342) compares.It is rabbit 83% that maximum homophylic polypeptide is arranged, and rat, people and mouse show 82%, 82% and 81% similarity respectively.On nucleotide level, pig CNTF is similar to people cDNA (88%) most, and the similarity of other species is rabbits 87%, rat 84% and mouse 84%.

The aminoacid sequence of aforementioned species and chicken (GenBank number of registration M80827) is arranged contrast and also is done.Pig CNTF is different from all five species 13 different positions.And the aminoacid sequence of pig four in seven other positions and described five species are inequality.According to MacVector ^TMGrouping, in all difference, seven positions show nonconservative amino acid change.These changes of pig and other species are: at 96, L-glutamic acid becomes Xie Ansuan; At 103, aspartic acid becomes glycine or L-Ala; At 132, Histidine becomes halfcystine, glutamine, or Serine; At 142, Threonine becomes methionine(Met) or L-glutamic acid; At 148, glycine becomes aspartic acid or arginine, and at 174, arginine becomes Histidine, and the position is 184, and L-Ala becomes Threonine, methionine(Met) or proline(Pro).If these positions play a crucial role on described proteinic function, the soluble heterologous protein of the difference of species obviously can not activate pig gp130 signal transduction pathway.At present the inventor is in and expresses in the bioactive flow of research of pig CNTF protein when determining it with allos CNTF comparison.

5 ' the promoter region of pig and people CNTF is by the existence of its possible transcription factor binding site point of extensive analysis.Several sites are found in two same positions between the species and guard, although the accurate sequence of concrete primitive is different; Yet other site is because in one of two species or the disappearance among both and slightly displacement.And, described 391bp district is just at upstream (the GenBank number of registration M63420 of the initiation site of people LiF, and analyzed whether Caenorhabditiselegans Osm-3 (GenBank number of registration D14968) contains the binding site of finding in pig and people CNTF promotor subunit J05436).

Guard in all analyzed promotor in these sites that attract people's attention especially, and comprising

activator albumen

1 and 2, several of AP-1 and AP-2 are in conjunction with primitive.These in conjunction with primitive are: an AP1_CS1 (STGACTMA), an AP-1_CS2 (TGAGTCAG), two AP-1_CS3 (TGANTMA), an AP-1_CS4 (TGASTMA), an AP-1-TRE-4/C (CTGAGTCAG) and three AP-2_CS6 (CCCMNSSS).AP-1 is generally expressed and by Jun, the dimer between Fos and the ATF family member constitutes, and AP-2 mainly expresses in neural ridge (Faisst and Meyer, 1992).AP-1 interacts with the primitive that contains TPA (12-neighbour-mnyristoyl-phorbol-13-acetate)-induction type enhancer element, known described enhancer element activated protein kinase C (Lee etc., 1987), and AP-2 transcribes inducing action by two kinds of different approaches mediations, described approach is phorbol ester activator enzyme C approach and cAMP deopendent protein kinase A approach (Imagawa etc., 1987).

Other site comprises three γ-IRE (CWKKANNY), they give the reactivity (Yang etc. to the lymphokine IFN-, 1990) and two binding sites (Nimer etc., 1990) of rHuGM-CSF GMCSF_CS (CATTW) (a kind of hemopoieticgrowth factor).And, a 30S-rRNA.I of potential (AGGT) binding site relates to initiation factor IF3-30s binding site (Ehresmann etc., 1986) and five TCF-1_CS sites (MAMMAG), they are to organize single-minded (Faisst and Meyer, 1992) to the T cell.Two sites in addition of only finding in the CNTF promotor are a α INF (AARKGA) binding sites, with an E2-A_CS site (RCAGNTG) factor-related with the E2-box, the E2-box factor may be myocyte or B cell-specific (Faisst and Meyer, 1992).Although the potential function in the several zones in the supposition conserved regions is can be attracting, releases bright its definite effect and remain transgenic analysis.

B. determine the chromosome position of pig CNTF by FISH

On pig Metaphase Chromosome smear, carry out apparent band of order R and FISH to determine the accurate chromosome position of pig CNTF gene.Description according to (1984) such as Ronne prepares the pig Metaphase Chromosome from whole blood.Substantially carry out FISH according to what (1993) such as Niebergs were described.In brief, contain the lambda particles phage clone that the 13kb genome inserts fragment (comprising complete CNTF gene) and be used as probe.By nick-translation, with digoxigenin-11-dUTP mark one microgram lambda bacteriophage dna.Tumor-necrosis factor glycoproteins in the described probe is suppressed by total pig genomic dna, and its method is that 70 ℃ of sex change 5 minutes are subsequently 37 ℃ of water-baths 30 minutes.

Described probe is transferred to by being immersed in freshly prepd 70% methane amide/2xSSC in cooled on ice and with the concentration of 10 nanograms/milliliter, and 70 ℃ continued 2 minutes and on the slide that contains the smear in mid-term of sex change.The fixed cap slide is also used the rubber dough closed edge on described slide.Described slide is incubated in 37 ℃ humidifying cell by temperature so that hybridization is carried out.By the anti-digoxigenin antibody of FITC link coupled, (Oncor, Gaithersburg MD) detect hybridization signal according to manufacturers instruction to use the Chromosomal in situ hybridization test kit.Redye karyomit(e) with iodate third ingot/anti-decolourant (Oncor), the apparent band of R is carried out in sealing in Hoechst 332581 anti-decolourants (Oncor).Also take pictures at Olympus Varox-T fluorescence microscope smear in mid-term with Kodak Ektachrome 100 chromatrope films.

95 smears in mid-term that contain hybridization signal have been analyzed altogether.Wherein, 27% (26) all show symmetrical hybridization signal at chromatid and karyomit(e); 20% (19) have hybridization signal on three right chromatids of homologous chromosomes; 40% (38) all show symmetrical hybridization signal on chromosomal two chromatids; And 13% (12) have a single signal on a chromatid.All the specific hybrid signal framing is with 6 subzones at the 2nd the short arm of a chromosome 1.The brachium ratio is calculated by 40 early metaphase karyomit(e)s, and wherein 32 (80%) shows that pig CNTF gene is positioned at karyomit(e) 2p1.6.Standard idiogram of this karyomit(e) that shows band FISH mark by R and described pig and R show and are with the 2nd karyomit(e) to arrange and contrast and be confirmed (tame pig stdn caryogram the council, 1988).

Embodiment 8

With the Oct-4 promotor and (or) enhanser expresses GFP

Be subjected to the expression of Oct-4 element regulation not to be subjected to the independent control of described promotor, and two enhansers that also are positioned at this upstream region of gene are controlled.One is called as the sexual cell specific enhancer and another is called the ectoderm specific enhancer.Use the wherein existing research of expression of two elements.

Described Oct-4 construction and CMV-GFP or PGK-GFP compare.Be significantly increased from the transgenosis colony number of Oct4 construction colony number than PGK and CMV construction.In addition, created from Oct4 enhancing subarea and " heterozygosis " construction of PGK promotor bonded.Described Oct-4/PGK-GFP heterocomplex produces the GFP more much higher than independent PGK-GFP and expresses.

Embodiment 9

The exploitation of vitro differentiation detection method

The external source of hematopoietic cytokine is supplied with and is done (ES) cell to be in a kind of undifferentiated state still be necessary keeping the mouse embryo.Confirm that recently hematopoietic cytokine utilizes the gp130 signal transduction pathway to keep this phenotype, yet they are not clear and definite as yet with the complex relationship of the versatility that keeps pig ES or PGC cell.Separation of pig ES cells in vitro and the differentiation of keeping because of not suppressing pig interior detail kytoplasm (pICM) are hindered.The optimal culture condition of described pICM is necessary.Therefore, the purpose of these researchs is will measure several allos hematopoietic cytokines and substratum to keep PGC cell or separated pig ICM in a kind of effectiveness of undifferentiated state.

The inventor has developed a points-scoring system with the variation in the differentiation state of the PGC cell of measuring vitro culture or described pICM.In first research, separate pig ICMs (the 7th day) and having or do not having human leukemia inhibitory factor (hLIF by the immunity operation; 1000 μ/milliliters) time, in based on the substratum (D/H substratum) of the substratum (D substratum) of DMEM or DMEM/Hams F-10 (1: 1), cultivated 4 days.In second research, as above collect pICMs and cultivation four days in six kinds one of are handled: control medium, human leukemia inhibitory factor (hLIF; 1000 μ/milliliters), human interleukin-6 (hIL-6; 100 nanograms/milliliter), hIL-6+hIL-6 soluble receptors (hIL6+sR; 100 nanograms/milliliter+2.5 mcg/ml), people's oncostatin M (hOSM; Or rat ciliary neurotrophic factor (rCNTF 10 nanograms/milliliter); 100 nanograms/milliliter).All cytokine prepares in the substratum of the improved Eagles substratum of Dulbecco/Ham F-10 (1: 1) for the basis.Described colony is taken a picture for morphological analysis usefulness every day.

Based on its morphology outward appearance pICM is divided into two types: A type (non-epithelial cell) or Type B (epithelial cell sample).Differentiation with standardized differentiation profile assessment pICM.With regard to each time point, each pICM series is divided into 1 (not differentiation fully) to 5 (differentiation fully) grade.By alkaline phosphatase activities, cytokeratin dyeing and scanning electronic microscope are confirmed differentiation.In first research, hLIF and substratum are not all postponed the differentiation (being respectively p=0.08 and p=0.25) of developmental pICM.In second research, at second day, the pICM that rCNTF cultivates was than the differentiation obviously low (2.07 ± 0.15 to 2.70 ± 0.16 of the pICM of hLIF cultivation; P＜0.05).And, add rCNTF and produce minimum ensemble average differentiation mark (2.53 ± 0.15).Yet, between 4 days by a definite date incubation period, do not have cytokine obviously to postpone differentiation (p＜0.05) with respect to contrast.Therefore, employed hierarchy system is to measure an effective tool of handling the effect of the differentiation of pICM in the growth.

Because these allos cytokines can not obviously suppress differentiation, they can not be favourable to separate pig ES cell under existing condition.The relevant homologous cell factor, the future studies of protein transformation period and dosage effect are provable to be more helpful.

Embodiment 10

The exploitation of atherosclerosis animal model

Coronary heart disease is considered to major causes of death in the U.S..From previous research, observe between the morbidity of the low-density lipoprotein (LDL) of rising and heart trouble much relations are arranged.Equally, the HDL in the high level circulation reduces relevant with the incidence of atherosclerosis risk.Therefore, as if HDL in the influence circulation and the E﹠H factor of LDL have entire effect to atherosclerotic sickness rate.Generally speaking, any environment of influence fat and cholesterol metabolic and transportation or hereditary effect expection will cause the ratio of lipoprotein levels in the circulation and the variation of ratio.As a result, be difficult to determine that concrete heredity or environmental effect and its are to the relation between may the influencing of coronary heart disease.

Proved that in the abiogenous sudden change of the mankind there is hereditary inducement in the cardiovascular disorder to some type.For example, described apoE allelotrope E2 and III type hyperlipoproteinemia (its sign is that triglyceride level and cholesterol levels rise) vitiligoidea and atherosclerosis have substantial connection.Equally, familial hypercholesterolemia is relevant with sudden change in the ldl receptor gene.Regrettably, the atherosclerotic sudden change case of not only certified influence quantity very little, and when identifying, the mutual relationship between genetic background and the environmental influence is difficult to study.This part is because restive environmental variance is difficult to carry out some Biochemistry Experiment with the mankind, and is difficult to carry out comprehensive genetic analysis (McCarrick etc., 1993) with the mankind.

On the other hand, the animal model of human diseases is allowed careful control and processing environment factor, allows in the different steps of described lysis and carries out detailed biological chemistry and Physiologic Studies, and can carry out analysis in the heredity more comprehensively than the mankind.Pig extensively has been used as atheroma and has been formed and thrombotic model, because it is in size, cardiovascular physiology and omnivory aspect and the mankind are seemingly.Dietary fat and cholesterol are carried out the comprehensive study that influences the aspect of Pigs Hearts popular name for morbidity, and its result has shown that pig is useful (Rapacz and Hasler-Rapacz, 1984) as the model of differentiating the factor that influences atherosclerosis formation.

In addition, pig provides unique model of understanding thrombosis and atherosclerosis process, because can implement careful operation and biochemical method easily.Therefore, pig has been used to use carotid artery vascular to form art and has studied relation between thrombotic degree and the blood vessel injury degree, effect by shearing resistance in the enhancement of using the reaction of external counterpulsation systematic study thrombosis, with by the effect (Fuster etc., 1991) of the cross transplantation carotid artery thrombotic model analysis and research Von Willenbrand factor in thrombosis and atheroma form.

In the past in 22 years, Rapacz, Hasler-Rapacz and colleague thereof have set up and have characterized in the pig detectable genetic polymorphism on the immunology of lipoprotein, and have set up and characterized these polymorphisms recently and be attended by relation between the atherosclerotic familial hypercholesterolemia.Demonstrate cholesterol by using the immunogenetics technology screening, the breeding animals of detectable quality and/or meristic variation in lipoprotein and the lipophorin, they have set up immune genetic project population (IPH).From 60 to 400 milligrams/deciliter of the plasma cholesterol variations that pig strain in this population shows.One of strain of being developed is characterized as being heredity blood plasma height-LDL and hypercholesterolemia (IHLC; Rapacz and Hasler-Rapacz, 1984), now be called hypercholesterolemia or familial hyperlipidemia (FH; Haler-Rapacz etc., 1995; Prescott etc., 1995; Rapacz and Hasler-Rapacz, 1989; Rapacz and Hasler-Rapacz, 1984; Rapacz etc., 1994).

In the recent period, along with the application of gene targeting in the ES cell, might produce the mouse muton (Piedrahita etc., 1992 that lack specificity lipophorin gene; Plump etc., 1992).Analysis to these animals has proved that this technology is useful to producing atherosclerosis animal model.One of muton that produces by the gene targeting in the ES cell is apoE deficient mice (Piedrahita etc., 1992; Plump etc., 1992; Zhang etc., 1992).The ApoE deficient mice has and is five times in the normal plasma cholesterol levels and is producing the abundant deposition of foam cell during age at its nearly aorta place to 3 moonrats.These spontaneous damages can develop and cause coronary occlusion (Plump etc., 1992 of serious coronary ostium in aged mouse; Zhang etc., 1992).Yet described mouse still can survive under the normal diet condition and reach (and surpassing) 1 year.Because their phenotypes that can survive and the carrying out property and the idiopathic result of their atheroma forming process, they have formed atherosclerotic useful animal model and should be useful when described disease incidence is exerted an influence helping to illustrate some diet and environmental factors.

According to experience with the apoE muton, the pig that defective apoE gene is carried in expection will produce and the apoE deficient mice in viewed similar phenotype; It is replenishing of the existing mouse muton of conduct not only, and itself help to illustrate some is difficult to study or do not have the atheroma formation of evidence in described mouse in meiofauna mechanism.In addition, by apoE locus (known can high frequency the locus of guiding) is studied, resulting animal model will be not only the atherosis good model of ripe prerolandic artery Rolando, and it is also important that the changing factor by removing the influence of the locus-specific of homologous recombination frequency makes successfully the chance of deactivation reach maximum.Along with the exploitation that versatility PGC derived cell system is arranged, and follow sign, now existingly may obtain apoE defective pig pig apoE gene.

A. the sign of transgenosis PGC derived cell developmental potency

For producing apoE defective pig strain, be necessary that any hereditary change that will be incorporated into the PGC derived cell passes to the next generation.No matter recently the report (Wilmut etc., 1997) of the adult organic developmental potency of the relevant adult body cell participation of portion how, and present method has several somatic advantages of use that surpass.At first, described PGCs has by mosaic and forms and consideration convey moves genetic modification is passed to follow-on potentiality.This is crucial advantage, because the consideration convey technology of moving does not only obtain excellent development and has the gained result possibility different with the result of sheep in pig, and somatocyte can not play a role to the formation of chimeric animal.Secondly, the homologous recombination ratio of somatocyte experience is than ES cell low (Arbones etc., 1994, Thyagaraja etc., 1996).Imagination EG cell and ES cell have similar guiding frequency, and cell of the present invention should be with the frequency direction higher than somatocyte.The 3rd, the efficient of consideration convey shifting method (even in sheep) uses the ectoderm of early embryo than using adult body cell height.Therefore, be successful if consideration convey moves, use and not break up the EG cell to move its efficient of donor as consideration convey should be higher.Therefore, described PGC derived cell has the advantage above the somatocyte method.

Therefore, can walk around to make in the mosaic to plant chance maximization that system transmits and whether mosaic formed and measure this two step, use the chimericization degree that produced as host's embryo with respect to the tetraploid of diploid embryos and the totipotency of described PGC derived cell is measured and move research by consideration convey.

1. genitaloid separation

Collect conceived pig uterus by hysterectomy, collection organization is for the usefulness of the DNA separation of parental generation analysis, and described tire is dissected, and separates sexual cell as described above (Labosky etc., 1994).Be to separate PGCs, shift out the sex-ridge of growing tire and temperature and incubate in the phosphate-buffered saline that contains 10mM EDTA (PBS) 20 minutes with the PGCs that dissociates from sex-ridge.After temperature was incubated, described ridge was punctured and the light and slow PGCs of discharging enters in the described substratum.Replenishing with the 0.01mM non-essential amino acid 2mM glutamine, the improved Eagle substratum of Dulbecco of 15% foetal calf serum (selected batch, Summit Biotechnology) and 0.1mM2 mercaptoethanol (PEG substratum): collect PGCs among the Ham ' F10.After the collection, wash 3 cells and be resuspended in the PEG substratum, wherein preferably contain soluble recombined human STEM CELL FACTOR 40 nanograms/milliliter, rh-bFGF 20 nanograms/milliliter and people LIF 20 nanograms/milliliter by centrifugal.

2. cultivate the PGC cell

Density is 10, the cell suspension of 000PGC/ milliliter by plating to as described above the preparation STO cell feeder layer on (Piedrahita etc., 1990).Cultivate after 7-10 days, have the morphologic colony of ES sample to be gone down to posterity new feeder layer to set up clone.The gained colony with 6-9 days interval by the tryptic digestion new feeder layer that gone down to posterity.The differentiation state of separated clone is measured (Moore and Piedrahita, 1996, Talbot etc., 1993) by the expression of morphology and alkaline phosphatase (a kind of mark that does not break up pig ICMs).

3. the blastocyst injection forms mosaic

The blastocyst injection technique is described as this paper substantially.In brief, pig PGC derived cell is dissociated into unicellular by tryptic digestion, and 12-15 transgenic cell is injected into the segmentation cavity of blastocyst stage embryo.After the injection, 10-15 indusium is transferred to each detected acceptor gestation and passes through the ultrasonography regular monitoring.Make animal gestation to amortization period and by the genomic dna engram analysis and outside determined chimericization degree by color and microsatellite marker quality testing survey transgenosis.

4. detecting kind of a system transmits

All being considered to chimeric animal is retained for kind of the usefulness of system's transmission detection.In brief, described chimeric animal and purebred Duroc animal mating, and its PGC derives genotype by identifying by color with to the distinctive microsatellite marker thing of described PGC derived cell outward.When utilizing transgenosis PGCs, analyze the transgenosis that exists among its offspring.When kind of a system taking place when transmitting, from this mating type and the offspring who comes is the purebred Duros with red pigments deposition characteristics.When the contribution of having only host's embryo was transmitted, its offspring was the hybrid that its peculiar patch appearance is arranged.At least 3 little sons are analyzed in every kind of potential mosaic.If not measuring kind is chimericization, then butchers described mosaic and obtain tissue to measure the scope and the distribution of PGC derived tissues.

5. produce PGC blastocyst mosaic with the tetraploid embryo

As described in (1996) such as Prather, produce the tetraploid embryo.Basically, two somatic embryos are to detect and post-coitum 52 hours collection during from the oviducal operation of hybrid (XB) gilt in rutting sedson.Embryo is equilibrated at the 0.3M mannitol to be added among the 5%HEPES buffering Tyrods (HbT) also with 5 volts/millimeter alternating-current fusion 10 seconds, 120 volts then/millimeters direct current 30 microseconds.After the fusion, embryo is placed in the Whitten substratum and cultivated 6 days at 39 ℃.

From transgenosis and the non-transgenic PGCs such as above-mentioned separated of purebred Durocs, cultivate, and injection enters the segmentation cavity of blastocyst stage embryo.Remove at 2 cell stages and collect diploid and cultivate 6 beyond the highest heavens, the tetraploid embryo that uses preparation as described above is as host's embryo.Behind 10-15 PGC derived cell of injection, indusium is transferred to the synchronization acceptor and is made and grows amortization period.By phenotypic markers, the chimericization degree and the type of offspring and its placenta tissue determined in microsatellite marker and karyotyping (XX:XY mosaic).With regard to phenotypic markers, the Duroc with its typical for red pigment is used as described PGC donor, and hybrid (Yorkshire x Hampshire; XB) gilt is used as host's embryo donor.Employed XB animal great majority are a little black patchess that have of white.Though because the existence of spot in the hybrid, this system can not be used to identify faint mosaic separately, when be used in combination with other mark or when chimericization by force to desired as a result the time as use tetraploid embryo, this system is useful.

With regard to microsatellite marker, several polymorphism marks have been screened.From the DNA sample of the parental generation of potential mosaic and described PGC donor and host's embryo by PCR as described above ^TM(Piedrahita etc., 1997) have been carried out analyzing.The existence of PGC-specific alleles shows that described PGCs participates in the growth of embryo among its offspring.By outer quilt and microsatellite marker be identified as chimeric animal fed for as above-mentioned detection kind system transmit.

Issuable potential problems are can not be conceived owing to the high mortality that shifts the back early embryo.Gilt generally needs the 3-4 tire to keep gestation in uterus.When using the tetraploid embryo to observe high not pregnancy rate, introduce the embryo conduct " carrier " that is not subject to processing.These extra embryos have increased the quantity of piglet in the described uterus, even so that only one of tetraploid embryo survival also can be cherished amortization period.By using XB, might identify the source of piglet, because the XB composition in experimental group contains the tetraploid caryogram and do not have injected PGCs can not proceed to amortization period as the carrier embryo.Therefore, the offspring of any Duroc of having sample phenotype derives from described injected cell.This phenotype is observed the confirmation that obtains genetic analysis as the aforementioned.

6. move the totipotency that detects the PGC derived cell by consideration convey

Use Willadsen (Willadsen, 1989) before to describe and by inventor's laboratory (Westhusin) and other people (Liu etc., 1995; Prather, etc., 1989) method revised carries out consideration convey and moves.In brief, collect ovocyte from the slaughterhouse or from the pig ovary that the jenny that undergos surgery for other purpose obtains.Described ovocyte is at external 39 ℃, contains the maturation described as (1996) such as Kim in the air of 5% carbonic acid gas 20 hours.Maturation medium is by replenishing with 10% pig follicle liquid, every milliliter 10 international unit horse chorionic-gonadotropin hormone (IntervetAmerica Inc., Millsboro, DE) and every milliliter 10 international unit hCG (Lypho MedInc., Rosemont, IL) NCSU23 of no BSA (Petters and Wells, 1993) forms.

Back 20 hours of ripe beginning, ovocyte is transferred to the new hole of maturation medium (500 microlitre) of no hormonal supplementation thing and cultivated other 20 hours.Cultivate when finishing, eddy current stirs with exposed ovocyte and brief contact 0.05% PRONASE A.Select the ovocyte of visible polar body and be placed on and contain in the Micro-Organism Culture Dish that replenishes with the TL Hepes substratum of 5 mcg/ml cytochalasin-B and 5 mcg/ml Hoechst33342 fluorescence dyes.The Micro-Organism Culture Dish that contains described ovocyte is placed on and keeps 37 ℃ and be fixed on the Zeiss stereoscopic microscope of being furnished with the Narshige micromanipulator on the stage of microscope of heating.Stoning pipette with an inclination when described ovocyte is maintained on the collection pipette by absorption removes polar body and the contiguous ovocyte tenuigenin of small portion.Confirm the stoning process of ovocyte by under uv irradiating, observing the tenuigenin of extracting out.

After stoning, briefly contacted 20 mcg/ml phytohemagglutinins to increase its viscosity as the PGCs of above-mentioned collection and genetic transformation, be placed on the enucleation oocyte perivitelline space and merge and transfer in the ovocyte tenuigenin by electricity.Fusion parameters is by single 1.6 kv/cm, and the electric pulse of 50 microseconds is formed.After electricity merged, described indusium was transferred in the new NCSU substratum of 500 microlitres and at 39 ℃, cultivates 6 days in 5% carbonic acid gas and the air.Blastocyst is transferred in the synchronization acceptor to produce the offspring.Detect and monitoring gestation by ultrasonic photograph.Lose when being accompanied by pregnancy loss when ultrasonic photograph shows a large amount of early embryos, move embryo transfer vector embryo with consideration convey as above-mentioned method.Move the piglet in research source from described consideration convey and identified easily, because they are Durocs of 100%.

When instructing from the nuclear of PGC derived cell when growing amortization period, any hereditary change of introducing described PGCs is carried very soon to of future generation and need not mosaic generate and plant system and detect (a kind of needs two generations method).

B. detect, analyze and keep the parameter optimization of transgenic pig PGC derived cell

Take two compensation processes.The first, identify that not only makes a tested transgenosis colony quantity maximization, and it is also important that the promotor that can play a cytodifferentiation state indicator.The second, identify the composition in the gp130 approach of pig PGCs and analyze them and when having the homologous cell factor to exist, keep effect among the PGC with multiplication culture.

1. detect the different promoters that control GFP expresses

The humanization GFP gene of use under CMV promotor control, observe during vitro differentiation that transgenosis is not broken up fluorescence very faint in the PGC derived cell and subsequently intensity increase.Therefore, to CMV-GFP and HSV-tk-GFP (HSV; Hsv), pgk-GFP (pgk; Phosphoglyceric kinase) and Oct-4-GFP bonded fluorescent signal study.Described HSV-tk and pgk promotor have been widely used in not breaking up modification (Koller and Smithies, 1992 of ES cell; Smithies, 1991), and described Oct-4 promotor is (Yeom etc., 1996) that function is arranged in ES cell and PGC cell.And described Oct-4 promotor is rapidly reticent (Yeom etc., 1996) when embryonic cell or ES cytodifferentiation in early days.

Described HSV and pgk all are cloned into pBS and described GFP is placed on the downstream of described promotor.GFP is cloned among the pBS of a modification and GFP is downcut by PstI digestion.Described then GFP is cloned among the plasmid pPGK that contains the pgk promotor in pBS.PPGK is by obtaining with PstI digestion pgk-neo box.These two fragments be joined together and by NotI digestion detection side to.Plasmid HSV-GFP makes up by separating the GFP/polyA fragment from CMV-GFP plasmid (obtaining from Steve Lacey).Described GFP is excised in the pBS of modified and by EcoRIV/SpeI digestion by subclone.Removing neo by BamHI/BglII digestion also self connects all the other plasmids and obtains the pHSV promotor from the HSV-neo box.Digest described pHSV with SacI then, fill and lead up end, and digest it with SpeI with Klenow.Two fragments are joined together to form HSV-GFP.Described promotor and structure gene all are 5 ' to 3 ' correct directions.

For obtaining the Oct-4 promotor, use PCR ^TMThe Oct-4 probe that produces screens a mouse genomic library and by restriction enzyme digestion and Southern analysis positive colony is mapped.The inventor separated and modified one contain with sexual cell and body early embryo cell in express the 3kb fragment (Yeom etc., 1996) of relevant promoter region.Described modification need be introduced an oligonucleotide that contains unique restriction site in the initiation site downstream.Introduce the initiator codon next-door neighbour of unique MluI site and Oct-4 by vitro mutagenesis.One provides many readings frame cloning site (MCS) of three possibility open reading frame (ORF) also to be introduced in the described MluI site.GFP is introduced into one of described cloning site and aligns fusion to read frame with Oct-4.

Linearized and introduce the described DNA of 5nM in the PGCs of quantity same as described above from the DNA of above-mentioned each construction by electroporation.The gained colony analyzes and measures the positive colony sum of transgenosis colony number/AP at electroporation after 7 days.Colony to 10 picked at random under the Olympus Vanox research microscope of being furnished with DIC and falling to penetrating the fluorescent visual parts is assisted carries out the digitized measurement strength of signal.With integrated Optronics DEI-750 high resolving power, low irradiation, the camera of 3-chip connects Neotech 24 bit color digitizing cards that are loaded in PoworMacintosh 8100 workstations and obtains gray scale image.Carry out Micro-photo densitometry and analysis with " NIH-image " or " Ultimate Pro " (Graftek company) software.The parametric statistic of strength of signal test difference is carried out with (Abacus) statistical packages.

2.gp130 approach and homology cytokine are to breeding and keep the effect of PGC derived cell

For increasing the efficient that goes down to posterity of pig PGC derived cell, the activation of having analyzed pig gp130 approach is to the propagation of PGC derived cell and the influence of survival.

A. illustrate the gp130 approach of pig

In mouse, multiple hematopoietic cytokine (CNTF, OSM, IL-11, IL-6+IlsR, and LIF) can be by activating the differentiation that the gp130 approach suppresses the ES cell.Reported similar result with new isolating PGCs and EG clone.This molecule family can pass through single-minded acceptor such as LIF acceptor (LIFR), CNTF acceptor (CNTFR), or solubility IL-6 acceptor (IL-6sR) works and induce homodimerization and heterodimerization (Yoshida etc., 1994) with gp130.And, having shown already that in fact IL-6 and CNTF were assembled into six aggressiveness mixtures, each contains two cytokine molecules, two α acceptors, a gp130 and a LIFR molecule or be respectively CNTF and two gp130 molecules (Deserio etc., 1995 of IL-6; Ward etc., 1995).Yet, the not existence of these acceptors of research or its information in any species outside mouse as yet so far.

For being increased in the efficient of passage in the phenotypic differentiation not, utilize nested RT-PCR ^TMCarry out CNTFR, LIFR, the information of IL-6sR and gp130 detects.This method is successfully made the gp130 pathway components that is used for illustrating people's embryo by (1995) such as Sharkey.Even the modifying method of this technology has also shown the information (Collins and Fleming.1995) of successfully amplifying from single mouse blastomere acquisition.

In brief, the PGC derived cell former generation colony in 0.05% trypsinase light and slow digestion 10 minutes with the described colony that dissociates from feeder layer.Draw colony respectively with mouth operation pipette, remove whole STOs, and (MRC, Cincinnati OH) gather in the crops total RNA, and described reagent is a kind of commercially available prod (Chomczynski and Sachi, 1987) that is similar to previously described sour phenol method with Tri-reagent.With oligo dT primer at 42 ℃ with the total RNA of AMV ThermoScript II reverse transcription (1 microgram)./ 10th of this reactant is used as the nested PCR of cDNA template to carry out describing as (1995) such as previous Sharkey ^TMTRAP.The acceptor of one or more these molecules exists prompting homology cytokine to activate pig gp130 approach and help long term maintenance PGC derived cell system.Be used to prepare the RT PCR of probe ^TMPrimer and the nested PCR that is used to detect the cytokine receptor expression ^TMPrimer is listed in following table 16 and 17.

Table 16

RT-PCR ^TMPrimer

SEQ???????????????????Tm??ProdGene????????????????????Primers?????????????ID????????Location???(℃)?Size

NOCNTF5′?????????GGATGGCTTTCGCAGAGCAAACAC????19?????????76-99CNTF3′?????????GCTGGTAGGCAAAGGCAGAAACTT????20????????444-420????61.2?378CNTFRexon95′???CGACCAGCACCACCAGCTC?????????21????????557-575CNTFRexon93′???CCAGGATGATGGGGACGCTG????????22????????676-657????55.5?120LIFR5′?????????CCAGTGGCAGTGGCTGTCATTGTT????23???????2705-2728LIFR3′?????????CCTGAGGTCTGTAACCCGCAGTTTT???24???????3272-3248???60.6?568GP1305′????????CCAAAGGACCTACTGTTCGGACAA????25???????1814-1837GP1303′????????CAGGACCGACTATGGCTTCAA???????26???????2128-2108???55.3?315OSM5′??????????TGCTCTGTGGATGAGAGGAACCATC???27????????627-651OSM3′??????????TTGCACCACCTGTCCTGATTTACAG???28???????1334-1310???59.0?708IL-65′?????????ATTCGGTACATCCTCGACGGCATC????29????????232-255IL-63′?????????TCGTCAGCAGGCTGGCATTTGT??????30????????595-574????61.4?364IL-6R5′????????ATCGGGCTGAACGGTCAAAG????????31???????1207-1226IL-6R3′????????AGCAACCAGGAATGTGGGCAGT??????32???????1547-1526???56.4?341OCT-4?exon15′??TCAAGGCTAGAGGGTGGGATTG??????33????????124-145OCT-4?exon13′??TCCAACCTGAGGTCCACAGTATG?????34????????449-427????55.0?326APOE5′?????????CAGTCCCTGTCTGACCAAGTGC??????35????????221-242APOE3′?????????TGCGGTAGAGCACCAAGCGG????????36????????458-439????55.5?238

Table 17

Nested RT-PCR ^TMPrimer

Gene????????????????????Primers?????????????SEQ?????Tm?????Prod.

ID????(℃)????Size

NOβ-Actin5′ext????GGGACATCAAGGAGAAGCTGTG?????????37β-Actin3′ext????ATGGAGTTGAAGGTAGTTTCGTGG???????38??????53?????215β-Actin5′int????TGGACTTCGAGCAGAGATGG???????????39β-Actin3′int????AGGATTCCATGCCCAGGAAG???????????40??????50?????151GP1305′ext???????CCAAAGGACCTACTGTTCGGACAA???????41GP1303′ext???????CAGGACCGACTATGGCTTCAA??????????42??????52?????315GP1305′int???????TCTTAGAGTGGGACCAACTTCCTG???????43GP1303′int???????CACCTTCATCTGTGTATGCTGCC????????44??????52?????192LIFR5′ext????????TGGCAGTGGCTGTCATTGTTGG?????????45LIFR3′ext????????GGAGGTGCATCTGTGGCTTATAGC???????46??????57?????490LIFR5′int????????GCTTGTGAGGGAAGCAGTGCTC?????????47LIFR3′int????????GGACGCTCAGCTACTGGGGA???????????48??????55?????137

B. homology cytokine expression

Prepared and be used for the pLIF that expresses at yeast and the artificial gene of pCNTF.These proteinic biologic activity detect as the description of (1996) such as Koshimizu basically.In brief, new isolating pig PGCs by plating have reorganization pLIF and (or) pCNTF and (or) on the deactivation feeder layer that exists of pOSM.Handle in triplicate.Described RT-PCR is depended in the selection of molecule ^TMThe result.Behind the plating 7-10 days, write down each positive colony number of AP in handling.Because the increase of colony number may be that propagation is all analyzed with apoptosis because the propagation of described cell or survival increase.

Use 5-bromo-2 '-Brdurd mark and detection kit to mix 5 bromines 2 '-Brdurd by the method for recommending and detect proliferation rate according to manufacturer (Boehringer-Mannheim).By two independently method detect apoptotic influence: use based on the ApoAlert symphysis albumen test kit (Clontech) that detects phosphatidylserine (a kind of early stage apoptotic indicator) at cell surface; Use is used to detect TUNEL (TdT-mediated dUTP nick end labeling-x breach end mark) dyeing of apoptosis characteristic dna fragmentation in late period.All three kinds of methods all have the advantage of energy quantitative analysis.Quantitatively based on colorimetric detection method or flow cytometer method.With regard to statistical analysis, the difference of every kind of detection to carry out in triplicate and to test to assess between the sample by Student t.

These results of study help to increase the PGC that obtains from one group of PGC cell derive colony number and help the described clone of long term maintenance.Two factor promotions move by the ability of the described apoE gene of homologous recombination deactivation with by mosaic formation or consideration convey described modification are transferred to the ability that described kind is.

The proof of C.PGC derived cell experience homologous recombination

Be the ability of proof pig PGCs experience homologous recombination, described GFP protein is directed to the Otc-4 gene.The expression pattern of Otc-4 is strictly limited to body early embryo cell and sexual cell (Yeom etc., 1996).In mouse, Oct-4 expresses (Yeom etc., 1996) in ES cell and EG cell and PGCs.And described gene is rapid inactivation between the differentiation phase of ES cell.

For making detectable guiding incident maximization, use a kind of gene trap method.In brief, described trap is by an acceptor splicing site (SA), and an internal ribosome entry site (IRES) and a no promotor GFP who has self polyA signal form.Correct insertion causes an exons 1-IRES-GFP bicistronic mRNA information, thus described GFP is placed on the control of Oct4 promotor down.Mountfourt etc. (1994) can be in mouse ES cells with no promotor beta galactosidase enzyme-IRES-Xin Meisu box described Oct-4 gene (IRES that leads; Internal ribosome entry site).When analyzing the G418 colony of survival, wherein experienced homologous recombination more than 80%.Therefore, use a kind of gene trap method and at the ES that grows, a locus enlivening of EG and PGCs phase causes the enrichment of homologous recombination.Can not grow under G418 selects because PRELIMINARY RESULTS has shown described PGC derived cell, described GFP gene is used as selected marker.Described construction is made up of 5 ' district's homologous fragment of 4kb and 3 ' district's homologous fragment of 4kb.

As before measuring in mouse, the homologous recombination frequency is subjected to the influence of homology degree between target DNA and the native gene.In mouse, this has not been a subject matter, because can be obtained easily from " isogenic " DNA that obtains with adorned the sort of identical clone.On the contrary, in pig, there is not isogenic kind to use.Therefore, the locus that is directed to is different from existing genomic clone probably, as inventors showing in the apoE locus.Can be in any heterology level that is directed to locus: a) use the NIH miniature pig by following former thereby reduce.This boar strain is developed from two pigs at first.Therefore, there are 4 allelotrope at most in each locus in this colony: b) use single boar generation all to be used for the PGCs that leads and study.If described genomic clone is isolating and be used to exploitation guiding construction from this boar, in at least 50% cell, will have and be present in the sort of " isogenic " allelotrope in the described guiding construction: c) owing to very be difficult to according to employed boar/strain isolated genes group clone repeatedly, long scope PCR ^TMCan be used to develop described guiding construction.In brief, from purpose strain/boar separated DNA will use produce by inventors or GenBank the long scope PCR of available sequence data design ^TMCondition and primer cover increase.In the situation of Oct-4 and apoE, inventors have separated and have checked order also contained the genomic clone of 8kb gene at least except that flanking sequence.This sequence data is used to design and detects the employed PCR of long range widening ^TMPrimer.

Long scope PCR ^TMBefore be used to obtain isogenic DNA to produce guiding construction (Randolph etc., 1996).This technology also can produce big amplicon (Barnes etc., 1994) to 35kb based on the reorganization of archaeal dna polymerase.The more important thing is the significant fidelity of this enzyme than homologous sequence length, error rate low in the 100kb 1bp.Therefore, the error rate of a conservative property will be that every 5-10kb has the 1bp mispairing.Low like this mispairing level should not influence described guiding frequency, proved as (1996) such as Randolph, they point out to lead frequency the construction of ordinary method (cloning of genome sequence) preparation with by long scope PCR ^TMThere is not difference between the oriented carrier of preparation.

Make the PCR of selection area ^TMPrimer, and 5 ' and the 3 ' homologous sequence and being incorporated in the cloning vector that contains promoterless Oct-4 gene of increasing respectively.As mentioned above, under the ideal amplification condition, might with high fidelity expand the zone that increases to 35kb length.In the present circumstance, the zone to the about 4kb of each homology arm is amplified.Therefore, this is just in described long scope method scope.

In case finish the Oct-4 construction, be introduced into Duroc and NIH miniature pig PGCs as described above.After plating and the cultivation, identify that fluorocyte and amplification are used for PCR ^TMAnd genome analysis.Use gene trap method, great majority insertion incident at random causes not having GFP and expresses.Therefore most of fluorocytes are directed to.Owing to insert at random and can not be identified that described target efficient is calculated as and is directed to the colony number than colony sum.

The result of this research helps to design apoE and rejects research because it provide a kind of to pig PGC derived cell experience homologous recombination ability and the indication of the relative efficiency of this method.Other gene that is used to gene trap method of expressing in PGCs comprises B2M, Rex-1, GAPDH, and Actin muscle.

D. make the apolipoprotein E gene inactivation by homologous recombination

By endogenous gene and the exogenous dna homology reorganization that is incorporated in the PGC derived cell, finish the apolipoprotein E gene inactivation.Pass through PCR ^TMBe the technology on basis, use isogenic DNA guiding construction and enrichment makes the maximized that detects a guiding incident with source event by the polyA trap.

Different with the oct-4 gene of high expression level in ES and the EG cell, in the expression that can not detect apoE in the PGC cell of not breaking up of mouse or pig.As a result, might utilize the used gene trap method of a kind of gene of Oct-4 as described.By uses (Danoff etc., 1997) such as Danoff, not too effective but still useful enriching method is used to lead the RANTES locus.In brief, this guiding construction comprises the homologous region of described target sequence, in the negative selected marker of a polyA under the control of himself promotor be connected to a diphtheria toxin (DT) under the terminal pgk promotor control of described construction 3 '.If described guiding construction inserts at random, then described selected marker or do not express or can not effective expression.Yet the homologous recombination incident causes this selected marker can utilize the polyA signal of target gene.

Therefore, for making the apoE gene inactivation, use a polyA trap method in conjunction with PCR and/or RT/PCR screening.In brief, described trap shows that by the apoE that is inserted in excalation is outer the negative GFP of a polyA under the control of oct-4 promotor in 2 forms.In addition, the apoE information that is introduced into to guarantee to stay of TAG termination signal is not translated.The insertion of correct guiding plasmid causes a bicistronic mRNA information that contains described GFP, mutant apoE and the many tails of this apoE.Make the unsuitable processing of GFP information owing to lacking a polyA signal, the detector efficiency of random integration incident is lower.The incident that is directed to is confirmed by PCR and/or RT-PCR.

This not only provides polyA for the suitable processing of described information and the maximum activity of selected marker, but also uses RT-PCR by allowing ^TMThat be directed to and the incident that be not directed to of discriminating and more convenient screening.In addition, described DT tail is that the homologous recombination incident should lack described tail and lose described DT gene because these incidents should mix the DT gene to the selection of random integration incident.The negative selective system similar (Piedrahita etc., 1992) of TK of this system and the mouse apoE gene that before had been used to lead.Described DT gene is used in this case, because it does not need to add 9-(1,3-dihydroxy-2-third oxygen methyl) guanine in substratum.DT gene before be used to by this way lead mouse genome and obtain kind of a system and transmit (McCarrick etc., 1993).

The deactivation of described apoE gene by native gene and be incorporated into Duroc and the exogenous DNA of NIH miniature pig PGCs between homologous recombination finish.This guiding incident comprises that the same gene with a copy replaces the complete apoE of endogenous, and described same gene is inactivated owing to the GFP gene inserts in its coding region.The guiding plasmid is introduced into PGCs by electroporation, and cultivates described cell 7-12 days.The fluorescence colony is amplified, and passes through RT-PCR ^TMWhether the DNA that analyzes them exists the diagnostic fragment of indication homologous recombination.In addition, from PCR ^TMPositive colony separated DNA with HindIII and (or) EcoRI digestion and by the agarose gel electrophoresis isolated fragment.After the separation, described dna fragmentation is transferred to nylon leaching film and seeks and visits the apoE gene of filter membrane.The existence of diagnostic fragment shows the guiding of described apoE gene.

Containing the allelic PGC derived cell of ruined apoE is used to move the generation offspring by mosaic formation or by consideration convey.Make whole transgenic progeny reach ripe and by breeding a test animal and analyzing transgenosis that its gestation exists and detect the allelic kind of ruined apoE system and transmit.This not only causes growing the apoE defective pig that can be used as valuable Atherosclerosis Model, and it is also important that it has proved the feasibility of carrying out gene targeting in pig.This causes using this technology aspect the exploitation of many biomedical researches field such as xenotransplantation and new gene therapy.

Embodiment 11

The totipotency of ox PGC derived cell

A. external and intravital embryo produces

Ox is fed on the pasture and replenishes to keeping the supplement feed that good physical appearance provides.For the embryo that obtains to produce in the body to be used for nuclear donor, twice intramuscular injection follicular stimulating hormone every day (FSH, Super Ov) totally 4 days, makes the super ovulation of cow.The FSH total amount of using does not wait (Yi Niu and decide) to 37 milligrams at 28 milligrams.(Kalamazoo is MI) so that the synchronization in oestrus for Lutalyse, Upjohn company to use 25 milligrams of prostaglandin(PG)s to Niu Yiqi when the 5th and the 6th injection FSH-P.Described ox is by artificial insemination, and after oestrus 5-7 days non-operation collection embryos.

With regard to external embryo produces, collect the ox ovary and be transported to the laboratory from local slaughterhouse, draw the immature egg parent cell from ovarian follicle there.The inspection ovarian follicle is drawn thing and is reclaimed described ovocyte and be positioned over fresh Tyrodes-Heps (TL Hepes) substratum (Bavister etc., 1993) under stereoscopic microscope.External oocyte maturation method is identical with the method that Crister etc. (1986) describes basically.In case isolate described ovocyte, just they are put into the 1 milliliter of tissue culture hole that contains 250 microlitre maturation medium, described substratum is by having replenished 10% foetal calf serum, 5 mcg/ml FSH, 5 mcg/ml LH (NCBL Inc., Sioux Center, IA) TCM199 with 1% penicillin streptomycin forms.Described ovocyte is placed on one 39 ℃, in the incubator of 5% carbonic acid gas and air 20 hours.For in vitro fertilization, melting chilling sperm, centrifugation and in discontinuous Percoll gradient (45%: 95%) with every milliliter 1.0 * 10 ⁶Cell is to mature oocyte artificial insemination (Parrish etc., 1986).After fertilization uses the description of BRL co-culture of cells system such as Voelkel and Hu (1992), at 37 ℃, and the described embryo of vitro culture in 5% carbonic acid gas and the air.

B. nuclear transplantation

With before being described by Willadsen (1989), by (1993b) such as Barnes, the method for modification such as Westjison etc. (1992) and Lavoir (1997) is carried out nuclear transplantation.Back 20 hours of ripe beginning, ovocyte is piled up cell by eddy current to eliminate.Select the ovocyte of visible polar body and put into the Micro-Organism Culture Dish that contains TL Hepes substratum and replenished 5 mcg/ml cytochalasin-B and 5 mcg/ml Hoechst33342 fluorescence dyes.The Micro-Organism Culture Dish that contains described ovocyte is placed on and maintains the temperature at 37 ℃ and be fixed on the Zeiss stereoscopic microscope of being furnished with the Narshige micromanipulator on the stage of microscope of heating.When described ovocyte is picked up by hand-held suction pipe, remove the ovocyte tenuigenin of polar body and sub-fraction vicinity with the stoning suction pipe of an inclination.Determine the stoning situation of ovocyte by the tenuigenin of under ultra violet lamp, observing sucking-off.

After the stoning, ovocyte is maintained in 37 ℃ the TL Hepes substratum.Incubated 4 minutes by temperature in 5 μ M inomycins about 24 hours of ripe back, succeeded by cultivate 3 hours activation enucleation oocytes (Lavoir etc., 1997) in 1.9mM dimethylamino-purine (DMAP).Perhaps, after stoning, make some ovocyte turn back to 37 ℃ of cultivations, activate and be used for consideration convey and move second day (ripe beginning back 38-42 hour) then.About 4 hours of activation back, PGCs (as above-mentioned collection and genetic transformation) briefly contacts the phytohemagglutinin of 20 mcg/ml to increase their viscosity, put into the enucleation oocyte perivitelline space, and transfer to (Westhsin etc., 1992) in the described ovocyte tenuigenin by the electricity fusion.In some cases, do the nuclear donor of contrast effect from the indusium usefulness in 5-6 days ages of ox collection or external generation.After electricity merges, described embryo such as the above-mentioned vitro culture of carrying out.Blastocyst is transferred and enters the synchronization recipient cattle to produce the offspring.Detect and monitoring gestation by ultrasonoscopy.

C. be used for the blastocyst injection that mosaic forms

The blastocyst injection technique is as described herein substantially.In brief, be dissociated into unicellular by tryptic digestion render transgenic ox PGC derived cell and 12-15 injection cell entered from the segmentation cavity of super ovulation ox is collected blastocyst stage embryo.After the injection, shift two embryos and detect and periodically monitor pregnant situation to each acceptor and by ultrasonoscopy.Make animal gestation measure chimeric degree to amortization period and by genome Southerns and microsatellite marker detection method.Screening and detection microsatellite marker (Piedrahita etc., 1997) under standard conditions.

D. the fusion parameters that is used for nuclear transplantation

The donorcells cytosome that Lavoir etc. (1997) report takes place to merge only is 45% to percentage ratio when using the tire ovogonium, and using blastomere by contrast then is 91%.This shows that it only is to merge with the electricity that PGCs carries out enucleation oocyte because lack suitable method that efficient reduces near 50%.Therefore, used different fusion parameters to enter enucleation oocyte to transmit PGCs.The unfertilized egg parent cell is by stoning and as above-mentioned activation.After making PGCs be inserted into described perivitelline space, described cell-cytosome is to one of in 8 processing that are assigned to 2 * 2 * 2 factor researchs.Fusion parameters is by 1 or 3 pulses, 1.5 or 2.0 kv/cm, and 25 or 50 microseconds are formed, and relevantly use the research of the ICM cell that is similar to the PGCs size to select according to inventors are previous.After the fusion treatment, described consideration convey moves embryo in external as above-mentioned cultivation.After 7 days, from cultivate, shift out embryo and assess its growth to blastocyst stage.

E. age and sex are to the totipotent influence of PGC-derived cell

When the growth of embryo/tire when carrying out, along with ovogonium entered meiophase at pregnant 65-80 days, become differentiation more and according to its heterosomal composition mode and alienation (Moens etc., 1996) of sexual cell.Although the PGCs phase is before ovogonium reduction division begins, before the divergence of some heredity and phenotype can occur in 65 days.This can influence the ability that PGCs instructs normal chick embryo to grow after nuclear transplantation or mosaic form.For studying this influence, from tire (group A, 2.0-3.0 centimetre (35-40 days) according to 3 difference pregnant ages of cronw rump definition, group B, 3.1-5.3 centimetre (45-54 days) and group C, centimetre 5.4-7.5 (55-65 days)) collect PGCs, transfection is also cultivated as described above.Every tire is operated respectively, and remaining tissue is used to DNA analysis to pass through PCR ^TMMeasure the sex of described tire.The nuclear transplantation method as mentioned above.

The nuclear transplantation embryonic development goes on record and by the nonparametric statistics analytical data to the per-cent of blastocyst stage in each is handled.In addition, blastocyst is transferred in the synchronization recipient cattle to compare pregnancy rate and to live young birth number.Make animal gestation to stopping and, carrying out genome Southerns method in skin and the muscle samples and measure genetically modified existence by at blood.

F. passage number is to the totipotent influence of PGC derived cell

Before derived with ICM and PGC derived cell system carries out studies show that morphocytology and (or) being expressed in that incubation time prolongs and changing (Piedrahita etc., 1990) during the passage number increase of marker.These variations can influence after the nuclear transplantation or injection enters clone developmental potentiality behind the blastocyst.For studying this influence, collect as described above, transfection, PGCs cultivates and goes down to posterity.Age and sex as tire donor as described in the above-mentioned selection.Collect the extracellular except that withholding from the the 5th, the 10 and the 20th, fresh cell is used to nuclear transplantation and blastocyst injection.Nuclear transplantation, the blastocyst injection, the embryo culture and the embryo that produce the calf that lives shift, data gathering, and the method for data analysis is all as mentioned above.Clone is measured as previously mentioned to offspring's contribution.

*????*????*

According to content disclosed herein, need not too much experiment and can prepare and implement open and claimed whole compositions and the method for this paper.Although the present composition and method are described with the preferred embodiment form, those skilled in the art should know to the change of described composition and method and to the step of method described herein or the change of sequence of steps and all not depart from notion of the present invention, aim and scope.More specifically, should know some reagent comprise the chemistry with physiology on relevant reagent can replace reagent described herein and obtain same or similar result.Those skilled in the art should know that all these similar substitutions and modifications all are considered to be in the defined the present invention's of claims aim, in scope and the notion.

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Sequence table

(1) physical data

(i) applicant:

(A) title: The Texas A ﹠amp; M University System

(B) street: 310 Wisenbaker

(C) city: College Station

(D) state: TX

(E) country: US

(F) postcode (ZIP): 77843-3369

(G) phone: (512) 418-3000

(H) fax: (713) 789-2679

(ii) invention exercise question: the composition and the method that produce transgenic animal species

(iii) sequence number: 51

(iv) computer-reader form:

(A) media type: floppy disk

(B) computer: IBM-compatible personal computer

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release #1.0, Version#l.30 (EPO)

(vi) application materials formerly:

(A) application number: US 60/027,338

(B) submission date: on October 11st, 1996

(v) application materials formerly:

(A) application number: US 60/046,094

(B) submission date: on May 9th, 1997

(2) sequence number: l data:

(i) sequence signature:

(A) length: 675 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(ix) characteristics:

(A) title/keyword: CDS

(B) position: 67..672

(xi) sequence description: SEQ ID NO:1:GCATGAACCT CTGAAAACTG CCGGCATCTA AGGTCTCCTT CAAGGCCCTC TGGAGTGCAG 60CCCATA ATG AAG GTC TTG GCG GCA GGA GTT GTG CCC TTG CTG CTG GTT 108

Met?Lys?Val?Leu?Ala?Ala?Gly?Val?Val?Pro?Leu?Leu?Leu?Val

1???????????????5??????????????????10CTC?CAC?TGG?AAA?CAC?GGG?GCC?GGG?AGC?CCC?CTT?CCC?ATC?ACC?CCG?GTC??????????156Leu?His?Trp?Lys?His?Gly?Ala?Gly?Ser?Pro?Leu?Pro?Ile?Thr?Pro?Val?15??????????????????20??????????????????25??????????????????30AAC?GCC?ACC?TGT?GCC?ACC?CGC?CAT?CCC?TGT?CCC?AGC?AAC?CTC?ATG?AAC??????????204Asn?Ala?Thr?Cys?Ala?Thr?Arg?His?Pro?Cys?Pro?Ser?Asn?Leu?Met?Asn

35??????????????????40??????????????????45CAG?ATC?AGA?AAC?CAG?CTG?GGA?CAA?CTC?AAC?AGC?AGT?GCC?AAC?AGC?CTC??????????252Gln?Ile?Arg?Asn?Gln?Leu?Gly?Gln?Leu?Asn?Ser?Ser?Ala?Asn?Ser?Leu

50??????????????????55??????????????????60TTT?ATC?CTC?TAT?TAC?ACG?GCC?CAG?GGG?GAG?CCC?TTC?CCC?AAC?AAC?CTG??????????300Phe?Ile?Leu?Tyr?Tyr?Thr?Ala?Gln?Gly?Glu?Pro?Phe?Pro?Asn?Asn?Leu

65??????????????????70??????????????????75GAC?AAG?CTG?TGC?AGC?CCC?AAC?GTG?ACT?GAC?TTC?CCG?CCC?TTC?CAC?GCC??????????348Asp?Lys?Leu?Cys?Ser?Pro?Asn?Val?Thr?Asp?Phe?Pro?Pro?Phe?His?Ala

80??????????????????85??????????????????90AAC?GGC?ACG?GAG?AAG?GCC?CGG?CTG?GTG?GAG?CTG?TAC?CGC?ATC?ATC?GCG??????????396Asn?Gly?Thr?Glu?Lys?Ala?Arg?Leu?Val?Glu?Leu?Tyr?Arg?Ile?Ile?Ala?95?????????????????100?????????????????105?????????????????110TAC?CTG?GGC?GCC?TCC?CTG?GGC?AAC?ATC?ACG?AGA?GAC?CAG?AAG?GTC?CTC??????????444Tyr?Leu?Gly?Ala?Ser?Leu?Gly?Asn?Ile?Thr?Arg?Asp?Gln?Lys?Val?Leu

115?????????????????120?????????????????125AAC?CCC?TAC?GCC?CAC?GGC?CTG?CAC?AGC?AAG?CTG?AGC?ACC?ACG?GCC?GAC??????????492Asn?Pro?Tyr?Ala?His?Gly?Leu?His?Ser?Lys?Leu?Ser?Thr?Thr?Ala?Asp

130?????????????????135?????????????????140GTC?CTG?CGG?GGT?CTG?CTC?AGC?AAC?GTG?CTC?TGC?CGC?TTG?TGC?AGC?AAG??????????540Val?Leu?Arg?Gly?Leu?Leu?Ser?Asn?Val?Leu?Cys?Arg?Leu?Cys?Ser?Lys

145?????????????????150?????????????????155TAC?CAC?GTG?AGC?CAC?GTG?GAC?GTG?ACC?TAC?GGC?CCC?GAC?ACC?TCG?GGC??????????588Tyr?His?Val?Ser?His?Val?Asp?Val?Thr?Tyr?Gly?Pro?Asp?Thr?Ser?Gly

160?????????????????165?????????????????170AAG?GAC?GTC?TTC?CAG?AAG?AAG?AAG?CTG?GGC?TGT?CAG?CTC?CTG?GGG?AAG??????????636Lys?Asp?Val?Phe?Gln?Lys?Lys?Lys?Leu?Gly?Cys?Gln?Leu?Leu?Gly?Lys175?????????????????180?????????????????185?????????????????190TAC?AAG?CAG?GTC?ATC?GCC?GTG?CTG?GCC?CAG?GCC?TTC?TAG??????????????????????675Tyr?Lys?Gln?Val?Ile?Ala?Val?Leu?Ala?Gln?Ala?Phe

195?????????????????200

(2) sequence number: 2 data:

(i) sequence signature:

(A) length: 202 amino acid

(B) type: amino acid

(D) topological framework: linear

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:2Met Lys Val Leu Ala Ala Gly Val Val Pro Leu Leu Leu Val Leu His 15 10 15Trp Lys His Gly Ala Gly Ser Pro Leu Pro Ile Thr Pro Val Asn Ala

20??????????????????25??????????????????30Thr?Cys?Ala?Thr?Arg?His?Pro?Cys?Pro?Ser?Asn?Leu?Met?Asn?Gln?Ile

35??????????????????40??????????????????45Arg?Asn?Gln?Leu?Gly?Gln?Leu?Asn?Ser?Ser?Ala?Asn?Ser?Leu?Phe?Ile

50??????????????????55??????????????????60Leu?Tyr?Tyr?Thr?Ala?Gln?Gly?Glu?Pro?Phe?Pro?Asn?Asn?Leu?Asp?Lys?65??????????????????70??????????????????75??????????????????80Leu?Cys?Ser?Pro?Asn?Val?Thr?Asp?Phe?Pro?Pro?Phe?His?Ala?Asn?Gly

85??????????????????90??????????????????95Thr?Glu?Lys?Ala?Arg?Leu?Val?Glu?Leu?Tyr?Arg?Ile?Ile?Ala?Tyr?Leu

100?????????????????105?????????????????110Gly?Ala?Ser?Leu?Gly?Asn?Ile?Thr?Arg?Asp?Gln?Lys?Val?Leu?Asn?Pro

115?????????????????120?????????????????125Tyr?Ala?His?Gly?Leu?His?Ser?Lys?Leu?Ser?Thr?Thr?Ala?Asp?Val?Leu

130?????????????????135?????????????????140Arg?Gly?Leu?Leu?Ser?Asn?Val?Leu?Cys?Arg?Leu?Cys?Ser?Lys?Tyr?His145?????????????????150?????????????????155?????????????????160Val?Ser?His?Val?Asp?Val?Thr?Tyr?Gly?Pro?Asp?Thr?Ser?Gly?Lys?Asp

165?????????????????170?????????????????175Val?Phe?Gln?Lys?Lys?Lys?Leu?Gly?Cys?Gln?Leu?Leu?Gly?Lys?Tyr?Lys

180?????????????????185?????????????????190Gln?Val?Ile?Ala?Val?Leu?Ala?Gln?Ala?Phe

195?????????????????200

(2) sequence number: 3 data:

(i) sequence signature:

(A) length: 603 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(ix) characteristics:

(A) title/keyword: CDS

(B) position: 1..600

(xi) sequence description: SEQ ID NO:3:ATG GCT TTT GCA GAG CAT TCA CCG CTG ACC CCT CAC CGC CGG GAC CTC 48Met Ala Phe Ala Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu 15 10 15TGT AGC CGC TCT ATC TGG CTA GCA AGG AAG ATT CGT TCA GAC CTG ACT 96Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr

20??????????????????25??????????????????30GCT?CTT?ATG?GAA?GCT?TAT?GTG?AAG?CAT?CAA?GGT?CTG?AAT?GAG?AAC?ATC?????????144Ala?Leu?Met?Glu?Ala?Tyr?Val?Lys?His?Gln?Gly?Leu?Asn?Glu?Asn?Ile

35??????????????????40??????????????????45AAC?CTG?GAC?TCT?GTG?GAT?GGT?GTG?CCA?ATG?GCA?AGC?ACT?GAT?CGA?TGG?????????192Asn?Leu?Asp?Ser?Val?Asp?Gly?Val?Pro?Met?Ala?Ser?Thr?Asp?Arg?Trp

50??????????????????55??????????????????60AGT?GAG?CTG?ACG?GAG?GCA?GAG?CGA?CTC?CAA?GAG?AAC?CTC?CGA?GCT?TAC?????????240Ser?Glu?Leu?Thr?Glu?Ala?Glu?Arg?Leu?Gln?Glu?Asn?Leu?Arg?Ala?Tyr?65??????????????????70??????????????????75??????????????????80CGT?ACC?TTC?CAT?GTT?ATG?TTG?GCC?AGG?CTG?TTA?GAA?GAC?CAG?CGG?GAA?????????288Arg?Thr?Phe?His?Val?Met?Leu?Ala?Arg?Leu?Leu?Glu?Asp?Gln?Arg?Glu

85??????????????????90??????????????????95CAT?TTT?ACT?CCA?GCT?GAA?GAT?GAC?TTC?CAT?CAA?GCA?ATA?CAC?ACC?ATT?????????336His?Phe?Thr?Pro?Ala?Glu?Asp?Asp?Phe?His?Gln?Ala?Ile?His?Thr?Ile

100?????????????????105?????????????????110GTC?CTC?CAA?GTC?GCT?GCC?TTT?GCT?TAC?CAG?CTG?GAA?GAA?TTA?ATG?GTG?????????384Val?Leu?Gln?Val?Ala?Ala?Phe?Ala?Tyr?Gln?Leu?Glu?Glu?Leu?Met?Val

115?????????????????120?????????????????125CTC?CTG?GAG?CAC?AAG?GTC?CCC?CCC?AGT?GAG?GCT?GAT?GGT?ACG?CCC?CTC?????????432Leu?Leu?Glu?His?Lys?Val?Pro?Pro?Ser?Glu?Ala?Asp?Gly?Thr?Pro?Leu

130?????????????????135?????????????????140AGC?GTT?GGA?GGT?GGT?GGT?CTC?TTT?GAG?AAG?AAG?CTG?TGG?GGC?CTG?AAG?????????480Ser?Val?Gly?Gly?Gly?Gly?Leu?Phe?Glu?Lys?Lys?Leu?Trp?Gly?Leu?Lys145?????????????????150?????????????????155?????????????????160GTG?CTG?CAA?GAG?CTT?TCA?CAG?TGG?ACA?GTG?AGG?TCC?ATC?CGT?GAC?CTT?????????528Val?Leu?Gln?Glu?Leu?Ser?Gln?Trp?Thr?Val?Arg?Ser?Ile?Arg?Asp?Leu

165?????????????????170?????????????????175CGA?GTC?ATC?TCC?TCT?CAT?CAG?GCT?GGG?GTC?CCA?GCA?CAC?GGG?AGC?CAT?????????576Arg?Val?Ile?Ser?Ser?His?Gln?Ala?Gly?Val?Pro?Ala?His?Gly?Ser?His

180?????????????????185?????????????????190CAT?GTC?GCT?AAG?GAC?AAG?AAA?ATG?TAG?????????????????????????????????????603His?Val?Ala?Lys?Asp?Lys?Lys?Met

195?????????????????200

(2) sequence number: 4 data:

(i) sequence signature:

(A) length: 200 amino acid

(B) type: amino acid

(D) topological framework: linear

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:4Met Ala Phe Ala Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu 15 10 15Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr

20??????????????????25??????????????????30Ala?Leu?Met?Glu?Ala?Tyr?Val?Lys?His?Gln?Gly?Leu?Asn?Glu?Asn?Ile

35??????????????????40??????????????????45Asn?Leu?Asp?Ser?Val?Asp?Gly?Val?Pro?Met?Ala?Ser?Thr?Asp?Arg?Trp

50??????????????????55??????????????????60Ser?Glu?Leu?Thr?Glu?Ala?Glu?Arg?Leu?Gln?Glu?Asn?Leu?Arg?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Arg?Thr?Phe?His?Val?Met?Leu?Ala?Arg?Leu?Leu?Glu?Asp?Gln?Arg?Glu

85??????????????????90??????????????????95His?Phe?Thr?Pro?Ala?Glu?Asp?Asp?Phe?His?Gln?Ala?Ile?His?Thr?Ile

100?????????????????105?????????????????110Val?Leu?Gln?Val?Ala?Ala?Phe?Ala?Tyr?Gln?Leu?Glu?Glu?Leu?Met?Val

115?????????????????120?????????????????125Leu?Leu?Glu?His?Lys?Val?Pro?Pro?Ser?Glu?Ala?Asp?Gly?Thr?Pro?Leu

130?????????????????135?????????????????140Ser?Val?Gly?Gly?Gly?Gly?Leu?Phe?Glu?Lys?Lys?Leu?Trp?Gly?Leu?Lys145?????????????????150?????????????????155?????????????????160Val?Leu?Gln?Glu?Leu?Ser?Gln?Trp?Thr?Val?Arg?Ser?Ile?Arg?Asp?Leu

165?????????????????170?????????????????175Arg?Val?Ile?Ser?Ser?His?Gln?Ala?Gly?Val?Pro?Ala?His?Gly?Ser?His

180?????????????????185?????????????????190His?Val?Ala?Lys?Asp?Lys?Lys?Met

195?????????????????200

(2) sequence number: 5 data:

(i) sequence signature:

(A) length: 1126 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(ix) characteristics:

(A) title/keyword: CDS

(B) position: 51..1001

(xi) sequence description: SEQ ID NO:5:GAGGAAGGAG GAGGAGGAAG CAACCGGGTT GGCCAATCGC AAGCCAGAAG ATG AGG 56

Met?Arg

1GTT?CTG?TGG?GTT?GCT?TTG?GTG?GTA?ACC?CTC?CTC?GCA?GGA?TGC?CGG?ACA????????104Val?Leu?Trp?Val?Ala?Leu?Val?Val?Thr?Leu?Leu?Ala?Gly?Cys?Arg?Thr

5??????????????????10??????????????????15GAG?GAC?GAG?CCG?GGG?CCG?CCG?CCG?GAG?GTG?CAC?GTG?TGG?TGG?GAG?GAG????????152Glu?Asp?Glu?Pro?Gly?Pro?Pro?Pro?Glu?Val?His?Val?Trp?Trp?Glu?Glu

20??????????????????25??????????????????30CCC?AAG?TGG?CAG?GGC?AGC?CAG?CCC?TGG?GAG?CAG?GCC?CTG?GGC?CGC?TTC????????200Pro?Lys?Trp?Gln?Gly?Ser?Gln?Pro?Trp?Glu?Gln?Ala?Leu?Gly?Arg?Phe?35??????????????????40??????????????????45??????????????????50TGG?GAT?TAC?CTG?CGC?TGG?GTG?CAG?TCC?CTG?TCT?GAC?CAA?GTG?CAG?GAG????????248Trp?Asp?Tyr?Leu?Arg?Trp?Val?Gln?Ser?Leu?Ser?Asp?Gln?Val?Gln?Glu

55??????????????????60??????????????????65GAG?CTG?CTC?AGC?ACC?AAG?GTC?ACC?CAG?GAA?CTG?ACG?GAG?CTG?ATA?GAG????????296Glu?Leu?Leu?Ser?Thr?Lys?Val?Thr?Gln?Glu?Leu?Thr?Glu?Leu?Ile?Glu

70??????????????????75??????????????????80GAG?AGC?ATG?AAG?GAG?GTG?AAG?GCC?TAC?CGC?GAG?GAG?CTG?GAG?GCG?CAG????????344Glu?Ser?Met?Lys?Glu?Val?Lys?Ala?Tyr?Arg?Glu?Glu?Leu?Glu?Ala?Gln

85??????????????????90??????????????????95CTG?GGC?CCC?GTG?ACC?CAG?GAG?ACG?CAG?GCG?CGC?CTG?TCC?AAG?GAG?CTG????????392Leu?Gly?Pro?Val?Thr?Gln?Glu?Thr?Gln?Ala?Arg?Leu?Ser?Lys?Glu?Leu

100?????????????????105?????????????????110CAG?GCG?GCG?CAG?GCC?CGC?GTG?GGC?GCC?GAC?ATG?GAG?GAC?GTG?CGC?AAC????????440Gln?Ala?Ala?Gln?Ala?Arg?Val?Gly?Ala?Asp?Met?Glu?Asp?Val?Arg?Asn115?????????????????120?????????????????125?????????????????130CGC?TTG?GTG?CTC?TAC?CGC?AGC?GAG?GTG?CAC?AAC?ATG?TTG?GGC?CAG?ACC????????488Arg?Leu?Val?Leu?Tyr?Arg?Ser?Glu?Val?His?Asn?Met?Leu?Gly?Gln?Thr

135?????????????????140?????????????????145ACC?GAG?GAG?CTG?CGG?AGC?CGC?CTG?GCT?TCC?CAC?CTG?CGC?AAG?CTG?CGC????????536Thr?Glu?Glu?Leu?Arg?Ser?Arg?Leu?Ala?Ser?His?Leu?Arg?Lys?Leu?Arg

150?????????????????155?????????????????160AAG?CGG?CTG?CTC?CGC?GAC?ACC?GAG?GAC?CTG?CAG?AAG?CGC?CTG?GCC?GTG????????584Lys?Arg?Leu?Leu?Arg?Asp?Thr?Glu?Asp?Leu?Gln?Lys?Arg?Leu?Ala?Val

165?????????????????170?????????????????175TAC?CAG?GCG?GGG?CTG?CGC?GAG?GGC?GCC?GAG?CGC?AGC?GTG?AGC?GCC?CTC????????632Tyr?Gln?Ala?Gly?Leu?Arg?Glu?Gly?Ala?Glu?Arg?Ser?Val?Ser?Ala?Leu

180?????????????????185?????????????????190CGC?GAG?CGC?CTC?GGG?CCC?CTG?GTG?GAG?CAG?GGC?CGA?TTG?CGC?GCC?GCC????????680Arg?Glu?Arg?Leu?Gly?Pro?Leu?Val?Glu?Gln?Gly?Arg?Leu?Arg?Ala?Ala195?????????????????200?????????????????205?????????????????210ACC?CTG?AGT?ACC?AGG?GCC?GGC?CAG?CCG?CTG?CGC?GAG?CGC?GCG?GAA?GCC????????728Thr?Leu?Ser?Thr?Arg?Ala?Gly?Gln?Pro?Leu?Arg?Glu?Arg?Ala?Glu?Ala

215?????????????????220?????????????????225TGG?GGC?CAG?AAG?CTG?CGC?GGA?CGG?CTG?GAG?GAG?ATG?GGC?AGC?CGG?ACC????????776Trp?Gly?Gln?Lys?Leu?Arg?Gly?Arg?Leu?Glu?Glu?Met?Gly?Ser?Arg?Thr

230?????????????????235?????????????????240CGC?GAC?CGC?CTG?GAT?GAG?ATG?CGT?GAG?CAG?CTG?GAG?GAG?GTG?CGC?ACC????????824Arg?Asp?Arg?Leu?Asp?Glu?Met?Arg?Glu?Gln?Leu?Glu?Glu?Val?Arg?Thr

245?????????????????250?????????????????255AAA?GTG?GAG?GAG?CAG?GGC?AGC?CAG?TTG?CGC?CTG?CAG?GCC?GAG?GGA?TTC????????872Lys?Val?Glu?Glu?Gln?Gly?Ser?Gln?Leu?Arg?Leu?Gln?Ala?Glu?Gly?Phe

260?????????????????265?????????????????270CAC?GCC?CTC?CTC?AAA?GGC?TGG?TTC?GAG?CCT?CTG?GTG?GAA?GAC?ATA?CGG????????920His?Ala?Leu?Leu?Lys?Gly?Trp?Phe?Glu?Pro?Leu?Val?Glu?Asp?Ile?Arg275?????????????????280?????????????????285?????????????????290CGC?CAG?TGG?GCC?GGG?CTG?GTG?GAG?AGG?ATG?CAG?TCG?GCC?GTG?AGC?ATA????????968Arg?Gln?Trp?Ala?Gly?Leu?Val?Glu?Arg?Met?Gln?Ser?Ala?Val?Ser?Ile

295?????????????????300?????????????????305AGC?TCC?TCC?ACC?TCT?GCG?CCC?AGT?GAT?AAT?CAG?TGAGTGCCCT?CTCATCCGGG?????1021Ser?Ser?Ser?Thr?Ser?Ala?Pro?Ser?Asp?Asn?Gln

310?????????????????315CACCCCCTTC?GGGGCCCCGT?TCCTGCCCAA?CTCCCCCGCC?TCCCCCAGCC?TTAGATGCCC?????1081TCTTGGTGGG?CCCCTGCTTA?ATAAAGATTC?ATCAAGCTTC?ACAGC?????????????????????1126

(2) sequence number: 6 data:

(i) sequence signature:

(A) length: 317 amino acid

(B) type: amino acid

(D) topological framework: linear

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:6Met Arg Val Leu Trp Val Ala Leu Val Val Thr Leu Leu Ala Gly Cys 15 10 15Arg Thr Glu Asp Glu Pro Gly Pro Pro Pro Glu Val His Val Trp Trp

20??????????????????25??????????????????30Glu?Glu?Pro?Lys?Trp?Gln?Gly?Ser?Gln?Pro?Trp?Glu?Gln?Ala?Leu?Gly

35??????????????????40??????????????????45Arg?Phe?Trp?Asp?Tyr?Leu?Arg?Trp?Val?Gln?Ser?Leu?Ser?Asp?Gln?Val

50??????????????????55??????????????????60Gln?Glu?Glu?Leu?Leu?Ser?Thr?Lys?Val?Thr?Gln?Glu?Leu?Thr?Glu?Leu?65??????????????????70??????????????????75??????????????????80Ile?Glu?Glu?Ser?Met?Lys?Glu?Val?Lys?Ala?Tyr?Arg?Glu?Glu?Leu?Glu

85??????????????????90??????????????????95Ala?Gln?Leu?Gly?Pro?Val?Thr?Gln?Glu?Thr?Gln?Ala?Arg?Leu?Ser?Lys

100?????????????????105?????????????????110Glu?Leu?Gln?Ala?Ala?Gln?Ala?Arg?Val?Gly?Ala?Asp?Met?Glu?Asp?Val

115?????????????????120?????????????????125Arg?Asn?Arg?Leu?Val?Leu?Tyr?Arg?Ser?Glu?Val?His?Asn?Met?Leu?Gly

130?????????????????135?????????????????140Gln?Thr?Thr?Glu?Glu?Leu?Arg?Ser?Arg?Leu?Ala?Ser?His?Leu?Arg?Lys145?????????????????150?????????????????155?????????????????160Leu?Arg?Lys?Arg?Leu?Leu?Arg?Asp?Thr?Glu?Asp?Leu?Gln?Lys?Arg?Leu

165?????????????????170?????????????????175Ala?Val?Tyr?Gln?Ala?Gly?Leu?Arg?Glu?Gly?Ala?Glu?Arg?Ser?Val?Ser

180?????????????????185?????????????????190Ala?Leu?Arg?Glu?Arg?Leu?Gly?Pro?Leu?Val?Glu?Gln?Gly?Arg?Leu?Arg

195?????????????????200?????????????????205Ala?Ala?Thr?Leu?Sar?Thr?Arg?Ala?Gly?Gln?Pro?Leu?Arg?Glu?Arg?Ala

210?????????????????215?????????????????220Glu?Ala?Trp?Gly?Gln?Lys?Leu?Arg?Gly?Arg?Leu?Glu?Glu?Met?Gly?Ser225?????????????????230?????????????????235?????????????????240Arg?Thr?Arg?Asp?Arg?Leu?Asp?Glu?Met?Arg?Glu?Gln?Leu?Glu?Glu?Val

245?????????????????250?????????????????255Arg?Thr?Lys?Val?Glu?Glu?Gln?Gly?Ser?Gln?Leu?Arg?Leu?Gln?Ala?Glu

260?????????????????265?????????????????270Gly?Phe?His?Ala?Leu?Leu?Lys?Gly?Trp?Phe?Glu?Pro?Leu?Val?Glu?Asp

275?????????????????280?????????????????285Ile?Arg?Arg?Gln?Trp?Ala?Gly?Leu?Val?Glu?Arg?Met?Gln?Ser?Ala?Val

290?????????????????295?????????????????300Ser?Ile?Ser?Ser?Ser?Thr?Ser?Ala?Pro?Ser?Asp?Asn?Gln305?????????????????310?????????????????315

(2) sequence number: 7 data:

(i) sequence signature:

(A) length: 671 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

( xi ) ：SEQ ID NO：7：AGATGTTGAC GTTGCAGACT TGGGTAGTCG CGAAGGTTTT GGCTGCTGGT GTTGTTCCAT 60TGTTGTTGGT TTTGCACTGG AAGCACGGTG CTGGTTCTCC ATTGTCTATC ACCCCAGTTA 120ACGCTACCTG TGCTACCAGA CACCCATGTC ACTCTAACTT GATGAACCAA ATCTTGAACC 180AATTGGCTCA CGTTAACTCT TCTGCTAACG CTTTGTTCAT CTTGTACTAC ACCGCTAACG 240GTGAACCATT CCCAAACAAC TTGGACAAGT TGTGTGGTCC AAACGTTACC ACCTTCCCAC 300CATTCCACGC TAACGGTACC GAAAAGGCTA GATTGGTTGA ATTGTACAGA ATCGCTTACT 360TGGGTGCTTC TTTGGGTAAC ATCACCAGAG ACCAAAGATC TTTGAACCCA GGTGCTGTTA 420ACTTGCACTC TAAGTTGAAC GCTACCGCTG ACTCTATGAG AGGTTTGTTG TCTAACGTTT 480TGTGTAGATT GTGTAACAAG TACCACGTTG CTCACGTTGA CGTTGCTTAC GGTCCAGACA 540CCTCTGGTAA GGACGTTTTC CAAAAGAAGA AGTTGGGTTG TCAATTGTTG GGTAAGTACA 600AGCAAGTTAT CTCTGTTTTG GCTAGAGCTC CATAATAGGG ATCCCAGAGC CTACTTTCAA 660GCCTGGAATC A 671

(2) sequence number: 8 data:

(i) sequence signature:

(A) length: 702 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

( xi ) ：SEQ ID NO：8：GGTGGTGCAG CTGCTTTCGC TGAACACTCT CCATTGACCC CACACAGAAG AGACTTGTGT 60TCTCGTTCTA TCTGGTTGGC TAGAAAGATC CGTTCTGACT TGACCGCTTT GATGGAAGCT 120TACGTTAAGC ACCAAGGTTT GAACGAAAAC ATCAACTTGG ACTCTGTTGA CGGTGTTCCA 180ATGGCTTCTA CCGATCGATG GGCTGCTGGA TCCGGTGGTA TCGATGGAGT GAGCTGACGG 240AGGCAGAGCG ACTCCAAGAG AACCTCCGAG CTTACCGTAC CTTCCATGTT ATGTTGGCCA 300GGCTGTTAGA AGACCAGCGG GAACATTTTA CTCCAGCTGA AGATGACTTC CATCAAGCAA 360TACACACCAT TGTCCTCCAA GTCGCTGCCT TTGCTTACCA GCTGGAAGAA TTAATGGTGC 420TCCTGGAGCA CAAGGTCCCC CCCAGTGAGG CTGATGGTAC GCCCCTCAGC GTTGGAGGTG 480GTGGTCTCTT TGAGAAGAAG CTGTGGGGCC TGAAGGTGCT GCAAGAGCTT TCACAGTGGA 540CAGTGAGGTC CATCCGTGAC CTTCGAGTCA TCTCCTCTCA TCAGGCTGGG GTCCCAGCAC 600ACGGGAGCCA TCATGTCGCT AAGGACAAGA AAATGTAGCA GTTACCTCCC TTCTTTCTTA 660GTTGCCTTCT ATTCTAATGG AATAGACAGT TCTCTGAGGG GG 702

(2) sequence number: 9 data:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:9:

TTGGTGTTCC?GAAAGCTGGC?TTCTG?25

(2) sequence number: 10 data:

(i) sequence signature:

(A) length: 26 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:10:

GGGTTATCAC?TGGCACTGGG?GGTGTA???26

(2) sequence number: 11 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:11:

AGCGACCAGC?CCAAGTGTAT?ACAG??24

(2) sequence number: 12 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:12:

GCCTGCTTTG?TCGTTCCTTC?AG?22

(2) sequence number: 13 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:13:

AGCGACCAGC?CCAAGTGTAT?ACAG??24

(2) sequence number: 14 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:14:

TGACCGCTTC?CTCGTGCTTT?AC?22

(2) sequence number: 15 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:15:

AGCTGCTCAG?CACCAAGGTC?AC?22

(2) sequence number: 16 data:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:16:

CTGAGGGTCC?AGACCACACG?G??21

(2) sequence number: 17 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:17:

GGATGGCTTT?CGCAGAGCAA?ACAC??24

(2) sequence number: 18 data:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:18:

GCTGGTAGGC?AAAGGCAGAA?ACTTG??25

(2) sequence number: 19 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:19:

GGATGGCTTT?CGCAGAGCAA?ACAC??24

(2) sequence number: 20 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:20:

GCTGGTAGGC?AAAGGCAGAA?ACTT??24

(2) sequence number: 21 data:

(i) sequence signature:

(A) length: 19 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:21:

CGACCAGCAC?CACCAGCTC????19

(2) sequence number: 22 data:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:22:

CCAGGATGAT?GGGGACGCTG?20

(2) sequence number: 23 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:23:

CCAGTGGCAG?TGGCTGTCAT?TGTT??24

(2) sequence number: 24 data:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:24:

CCTGAGGTCT?GTAACCCGCA?GTTTT?25

(2) sequence number: 25 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:25:

CCAAAGGACC?TACTGTTCGG?ACAA??24

(2) sequence number: 26 data:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:26:

CAGGACCGAC?TATGGCTTCA?A????21

(2) sequence number: 27 data:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:27:

TGCTCTGTGG?ATGAGAGGAA?CCATC?25

(2) sequence number: 28 data:

(i) sequence signature:

(A) length: 25 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:28:

TTGCACCACC?TGTCCTGATT?TACAG?25

(2) sequence number: 29 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:29:

ATTCGGTACA?TCCTCGACGG?CATC??24

(2) sequence number: 30 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:30:

TCGTCAGCAG?GCTGGCATTT?GT?22

(2) sequence number: 31 data:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:31:

ATCGGGCTGA?ACGGTCAAAG?20

(2) sequence number: 32 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:32:

AGCAACCAGG?AATGTGGGCA?GT?22

(2) sequence number: 33 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:33:

TCAAGGCTAG?AGGGTGGGAT?TG?22

(2) sequence number: 34 data:

(i) sequence signature:

(A) length: 23 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:34:

TCCACCTGA?GGTCCACAGT?ATG?23

(2) sequence number: 35 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:35:

CAGTCCCTGT?CTGACCAAGT?GC?22

(2) sequence number: 36 data:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:36:

TGCGGTAGAG?CACCAAGCGG?20

(2) sequence number: 37 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:37:

GGGACATCAA?GGAGAAGCTG?TG?22

(2) sequence number: 38 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:38:

ATGGAGTTGA?AGGTAGTTTC?GTGG??24

(2) sequence number: 39 data:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:39:

TGGACTTCGA?GCAGCGCTGG?20

(2) sequence number: 40 data:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:40:

AGGATTCCAT?GCCCAGGAAG?20

(2) sequence number: 41 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:41:

CCAAAGGACC?TACTGTTCGG?ACAA?24

(2) sequence number: 42 data:

(i) sequence signature:

(A) length: 21 base pairs

(B) type: nucleic acid

(E) chain: strand

(F) topological framework: linear

(xi) sequence description: SEQ ID NO:42:

CAGGACCGAC?TATGGCTTCA?A????21

(2) sequence number: 43 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(G) chain: strand

(H) topological framework: linear

(xi) sequence description: SEQ ID NO:43:

TCTTAGAGTG?GGACCAACTT?CCTG??24

(2) sequence number: 44 data:

(i) sequence signature:

(A) length: 23 base pairs

(B) type: nucleic acid

(I) chain: strand

(J) topological framework: linear

(xi) sequence description: SEQ ID NO:44:

CACCTTCATC?TGTGTATGCT?GCC??????23

(2) sequence number: 45 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(K) chain: strand

(L) topological framework: linear

(xi) sequence description: SEQ ID NO:45:

TGGCAGTGGC?TGTCATTGTT?GG?22

(2) sequence number: 46 data:

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(M) chain: strand

(N) topological framework: linear

(xi) sequence description: SEQ ID NO:46:

GGAGGTGCAT?CTGTGGCTTA?TAGC?24

(2) sequence number: 47 data:

(i) sequence signature:

(A) length: 22 base pairs

(B) type: nucleic acid

(O) chain: strand

(P) topological framework: linear

(xi) sequence description: SEQ ID NO:47:

GCTTGTGAGG?GAAGCAGTGC?TC?22

(2) sequence number: 48 data:

(i) sequence signature:

(A) length: 20 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi) sequence description: SEQ ID NO:48:

GGACGCTCAG?CTACTGGGGA?20

(2) sequence number: 49 data:

(i) sequence signature:

(A) length: 4791 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(xi)：SEQ ID NO：49：GGTACCCCTG GCAATTTTAG GGGCAGTGTG GTCTCAGATA TGTGCCTGGC TGGCACTCCC 60CCACCTCTGT TTTTTCTTTT TTGAATGCTT TTTAATGGGA GGGGATGCCA AGCTCATTCT 120CCCTAGTCCT CACCATTCCT TCCTGCCCCT CATTCATTTC TGAACCTACC TGCCGCCAAA 180GACACCAGAA TCTGTGACTC TGCCTAAGAG GCTCCGGAAG TGCCTTTAGG GGGCCATCAC 240CCTGCTCTGA GCCCCAAGGG CCCTCCAAGC CGCTTCTGTT CTCCCCAACC ATCTCTCCGC 300ATCCCAAGGA GCAAGCCCAG GTTCAGTTGG CTAAGTCTCT GCTTCAAATG TCCTTTCAAC 360ATGGAAATGC AGGACCACCC TCCCTGGAAG GGAGCCGAGA GATCAGGCAA ACGCCAGACG 420CACCACACTT TCCCCGAAGC TCCTCCCCAG CGAAGGCAGT CACAGCAGCC CCTCAGCTTT 480CATCTTGCCA GCCCCTCCTG GGCCCCACCG GTGGTCAGCA GTGCCGGCTT GCTGATGGCG 540CTCAGGAGGC CTGACCGTGT GGGACCCTGG GCAACCTGTC CTCTTGGGAG CCCCGCTTTG 600CCCGGTGGCA GCGGGCAGGA TGGAGGAGGC CATCCCTGTG GTGGCTTCCA GGGCCCACGT 660TCAGTTTCGC TTTATTGCCG TCTAAAGTCC ACGCCAGGGC CCCAGCCCCT CTTCCTGGAA 720CCTCCTTCTC AGCCAAGCCC CAGCCTGCCT CCTCCCCACT GCCCCCCTCC AAACCCCTTG 780ACCAGGGGGC TGCAGGTGTC AGCTCCCATC TCCTTGCCTA GTTTCAAGCC ACCTCCCCAA 840ACCCAGGTGA GTCAGGGTTG TCTGGGGTGC CCACTTCCCC GGGGGAGGGG GGACCCTGAT 900GACTCGGGGA CCCTCCCTCC CCCCCATCTT CAAACAACTC CCAGGGCAAG CCGCTCAGGA 960AAACCGCAGG GTGTTTTGTT GAAGAGTTCA TTATAATTTT ATCAATCAAA TTCTTAGAAG 1020AGGGAAAAAG TCTGCTCTCC CCACCCTCCC CCCTCACTGC CCCCCCTCCA CTCTCACTTT 1080CTTCCATTCA TAATTTCCTA TGATGCACCT CAAACAACTT CCTGGACCGG GGATCCCTGC 1140TAAATATAGC TGTTTCTCTC TCTGTCTTAC AACACAGGCT CCAGTATATA AATCAGGCAA 1200ATTCCCCATT TGAGCATGAA CCTCTGAAAA CTGCCGGCAT CTAAGGTCTC CTTCAAGGCC 1260CTCTGGAGTG CAGCCCATAA TGAAGGTCTT GGCGGCAGGT AAATCCACCC GCCCCCTGCC 1320CCGGGCTGGC TTCCGCCGGG AGCCCCGGCC GCGGGCGCCG CGGCAAACTT GGGGCCCCTG 1380GCGATCGCGA GCGGGACACC CACCCGCCGC AGACACACGG ACACTTGGGG CGCCCGCGCA 1440GCCGAGAGCC CGGGCCGCCG GAGGCGCCGG GTGGCGGCCG CCGCCGGGGG CGAGCGCGGG 1500CACATGGTCC CCGCACCTCG CGCCCAGCCC GCCCGGGGCC CCGCAGGTTG GCGGGCCAGG 1560CAGGGGGCGC CGCTGCCTTC CTCCTCCTCC TCCTCGCTCC TGAACTCTCG CCGCTGCTCT 1620TCCCGCTCAG CTTTGTCCGG GATTCACCCT CCCTCTTTTC TTTCTTTTTC TTTCTTTCCG 1680CTTTCTTTTC CAACCGCGTC CCCGGCTGCT CCCTGGGAGG GGCGCCGGCC GCCGGAGCAG 1740CTCGCAAACT CCGGCCCGGG ACGGGAGCAG GTGCCGCCTC CATCTGCTAG AGCCCGGAAA 1800GCTGTGGTCT GTGCTAGGTG AGCCCGGGGT GTGGGGCACC CCCCGCCCCC CCGCCAGCCA 1860TCCTGGGGCC TGAGCCCTGC CTGGAGATGC TGGGAGGCAC AGGGGACCCA GAAGTGAAGT 1920CGAGGCTGCA CTGTCCCAGC CGAGGAACGG GCTCCAGAGC GCCTCCCCTT CCTCCAGTCT 1980CCTCGCTTCT CCAACTCTCA CAGGTCCCCC GACCCCAGCC CTTGCTGCAG GGTCATCTGG 2040AACACAGAGA GGGGTGGGTG GCAAGCAGGG CCCCCCTGCC CTCCCTGCGG GGAGGGGTGC 2100TCCTGGACAG GCCTGGACAG ATCTCCCCTC TCCCTCTCAC CTTTCACTTC CCTCCCTCCC 2160CCGCCCACCT GGCTGCCTGC AACCTTTTCC CTTTTTTTCT TTCCTGGTTG CACCATTCCC 2220TCTCCCTCTT GAAGGCTCTG AGGGCGTCTG TGGAACCCCA GGATTCTCCT TGTCCTAACC 2280CGCAACCTGG GACGAGGACC AGTGAACAAG AGTGTGGGGG GTGGGGGGTG GAGCTCGGAG 2340GCCAGGAGCA GCAAGGAGCC AGGAAGGGAG GTTCTGAAGG ATGCCCTGGC ACTGGAGAAG 2400GGGGCAGAGT TGCAGCCCTG GGGTGGAGTC CAGGGTCCCA GAGAGGGGAC TGGCCACATC 2460TGGAGGAGGA GGAGCACAGA GAAGCTGGGG AAAGGTGACA GGATCAGGGG GAAAAAGGCC 2520CAGTGAGCCC ACATCACCGA GACAAGTTTG GGAGATGAGG GTCAGCAGAA ACCCCCGCTC 2580CCCGTGGGCC TGGTGGGAGC CCACTCTGTG AGACAGGAGC ATGAAGTAAC ACTTAGGAAT 2640CTGGACCTTC CTGGGGAGTT AAGGATCTTT TTCTTGAGAC CTGGGGCATC GTCCCCTCCT 2700GGCAGAGGCC TGGAGGGTTG GTATCACTCT GAATCCGGTT CTCAGCTGAT AGGAACAGCT 2760CATGTCCTGT GCCCCTTGGT CCCCCCAGGA GACAGCAGGG AGTGATAAAC AGGGAGATTT 2820AGCCATCTGG GGAGGTAGAT GCAGGGACAT TGCGAAAATC AGAAACCGCC AGGTCTGGAG 2880AAGAGAGAGC TGGAGCCTGA GAGGGGAACG TCCCTGCAGG ACCAGAGTCG CAGCCTCTCC 2940CCTAAGCTGC TTGCCCGCTG CCCCCCACCC CGCCACCCCT GCTCATGGCT CCCCACCGCT 3000TGTCTGCAGG AGTTGTGCCC TTGCTGCTGG TTCTCCACTG GAAACACGGG GCCGGGAGCC 3060CCCTTCCCAT CACCCCGGTC AACGCCACCT GTGCCACCCG CCATCCCTGT CCCAGCAACC 3120TCATGAACCA GATCAGAAAC CAGCTGGGAC AACTCAACAG CAGTGCCAAC AGCCTCTTTA 3180TCCTCTATGT AAGCCTCCCC CTCAGGGTAC CGAGGAACAG GCAGGGAGGG CTGGGGTCTG 3240CAAGCAGGGA CCTGGGCTGG TGCGGCTGGT CAGAGAAGGG AATGGTGTGT GGTTTTGTCC 3300CACTGCACCC AGATCCCCCC AGCTTCCTCC CCATCCCGCG GCCGAGGCCC GGCCTCTTTC 3360CCTTGGTGCC AAGGTAGATG GGGCGGGGGG GGGGGAGGCG GGCAGAGGCT CCTGGAGAAG 3420AGGTGGCAGG CAGGGCTGGC ACTTGTAGCA TTGGGATTTG TCCACCTGGT GGAGGGAGGC 3480AGATGACAGA GAGGGAGGCG GTGGAAGGGA CTGGGGAGGT GCTGTTGAAA GAGACAGCGG 3540GCTGTGGGTG ACGGGGTGCG GAGCCGCCCA GGAAGAGGGT GAATGCGGTG GGTGAAAGGG 3600CAAGTGTGTG TGGTGTACAA GGCTGGAGGT GAGACTGGGT GTTTCCCCCC CTCTCCCTTG 3660TGGTCCTGAT GCGGGTGATG AGGAGGGTAC CTCCTTGCGT GGGATAGAGG CTGGATGCTT 3720TAGCAAGTGC ATTCGCGCCC ACTGCTACTT CTGGCTCTCG GGACAGTCCC GAGATGCCTG 3780CAGGGCAAGT GATTGGATTC TCAAGCCCCT GTGTGTGTGT GTGTGTGTGT GTGTGTATTG 3840TGGGGGGGCG GCACTGACGC CCAAGGGCTG ACCACAGGCG GGGCAGCAGG GCTGGAGCAG 3900CCGTCCCTGC CTCCCACTTC ACCACCCCTC TGCCCCTCTG CTCCTCAGTA CACGGCCCAG 3960GGGGAGCCCT TCCCCAACAA CCTGGACAAG CTGTGCAGCC CCAACGTGAC TGACTTCCCG 4020CCCTTCCACG CCAACGGCAC GGAGAAGGCC CGGCTGGTGG AGCTGTACCG CATCATCGCG 4080TACCTGGGCG CCTCCCTGGG CAACATCACG AGAGACCAGA AGGTCCTCAA CCCCTACGCC 4140CACGGCCTGC ACAGCAAGCT GAGCACCACG GCCGACGTCC TGCGGGGTCT GCTCAGCAAC 4200GTGCTCTGCC GCTTGTGCAG CAAGTACCAC GTGAGCCACG TGGACGTGAC CTACGGCCCC 4260GACACCTCGG GCAAGGACGT CTTCCAGAAG AAGAAGCTGG GCTGTCAGCT CCTGGGGAAG 4320TACAAGCAGG TCATCGCCGT GCTGGCCCAG GCCTTCTAGA CGGGAGGTCT TAGATAGTAG 4380GGGACTCTCC AACTGCAGCC GTGGCCCAGA GCACTGCCAG ACCCGAGTAG GGGCCGCTGG 4440CAGACCCCTG AGGGGGTTCC TGGCCGGTCC ACTCCCCTCC AGGGTGGGCC GCCACGAAGC 4500CGAGCAGAGC CAGAACTCCC AGAGGCAGAA CCTATACGTG GTGCCAACTA GAAAGGAAGG 4560CGCCCCTTCT TCTGGGAGAC TACAGCCGGG CACGCAGTGT CGGGCTGGAG TTTGGCCCCT 4620GACTCATCCC CTCAGCCAGG GTCTTTGTGA GCAAACCCCG AAAGTTGTCT CTGGCGACCC 4680TGACCACGGG GTGAGACAGC AGGGGTCGGG GGCACTAACC CGCGACCCCC CAGCAGAATG 4740ACCACCATCA GTGCCTTGGC TGACCTTGAA AGGTCTGGTT GGAGCTCCAG C 4791

(2) sequence number: 50 data:

(i) sequence signature:

(A) length: 2680 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

(ix) characteristics:

(A) title/keyword: modified_base

(B) position: one of (1000,1064)

(D) other data :/note=" N=A, C, T or G "

( xi ) ：SEQ ID NO：50：TAATAAAATG AACGGATTAA ATGTGTATAT AAAATAGATT ACATTAGGCA AGGGTGTCAT 60GGGATTAGTG TGACAGTGAC CTTGTTGCTA AGAGCAAAAG CAAAACAATG ACTTAAAAAC 120AAGTACACTA GGCACTGAGT GGAGGAGAGA GATGGAGGCA GACGCTACAG GAAAAAAGCT 180GATTAAAAAG GGGCCTTTGA TTCCACAGGC ACAAAAATCC ACAGCCAGGA ATTTGCTGCC 240ACCTCTGAGT CAGGCAGGGG GTGGGGGTGC ACAATTCCAT TAGTAGAGAA ATGCCCAGTG 300GATTTAGTCT GAGAGTCACA TTGCTTATTT GGACCAGTAT AGACAGAAAC AAACCCAGCT 360CACTTGTTTC CTGGGACAGT TGAGTTAGGG GATGGCTTTT GCAGAGCATT CACCGCTGAC 420CCCTCACCGC CGGGACCTCT GTAGCCGCTC TATCTGGCTA GCAAGGAAGA TTCGTTCAGA 480CCTGACTGCT CTTATGGAAG CTTATGTAAG TTGCCTGTTT TCCTGTTGTG TCTTTTCACC 540TCACTTCTTC TGATCCAGCC CCTTACCATC ATGCTTCAGG CCGTTACCAG CTATGCAAGA 600CCTAACCATA CAGTCATTTG TATATTGGGC ACCTATCATT TGACATGGCC CCTTCCCTTG 660AGGAAACTCA TGAGACCTTA TTTTCTTTCT GTAGGCTATC TAGGAGACAT GTTCCTTGAA 720TAAGAAAATA CAGGCTTCTA GATAGAATTG GTTATATATC TTGGAGGCTG TTCTTAATAT 780GATTACCTTA TATGATTACC TTAATATGAT TACCTACGAG TCCTAGTTCT ACCAGAATAC 840ATTAACCATA TTTGGGATCT TCGTGTACAT TGTTGTGATT TTTTGAGCTG GTAAATGAAA 900AGCAGAGTGA GGTTTATAGG ACTGAGAGAA CAGTATAAAC CCAACGAGTT TCTCCTATAT 960GGTATAAGCA TCTGTGTATG AATTACAATC AAAAGTGTTN CCCTGTGTCT AAATAGAAAG 1020GTAACCTACA CTGCCAAAAA AAAAAAGAAA AAAGCCATAG AAGNATCACT GGGGACTTGA 1080GGAAGTGTCA GATTCAGATA GGTTTTCTGA TAGAAGAATA TTCCCAACAG TCTTTACCTA 1140AGGCCTGTCA TGGAAACACT CCAGGCTCCT GTAGAGAGTT CTGATTTAGG TTCTTTATGA 1200ACTAATTTAT CTTCATATAG CCCTACTAGT CAGAAATCAC ACTCTTCAAA ATACCAATTT 1260TTAAAAATAA TTTCCATTGA ATTCTCCAAT AAAGGATTGT CCTTACCATT GAAAGTGGGC 1320AATGGAGCAG AGAAAAATTG GAAAAATTCT ATGATGGCTA TATTCTAGGG CTTCCCAGGT 1380GTAGGGCTTC CCAGTGTCTT CAGGGGTATT TAAAATGTGT AGACTCCAGT ATCATTATAC 1440TATTCCAGTT TCCAGGAGGT GTTTCAAATA GGAAGGAAAG ATTATTCTAG GCCAGTCAGT 1500GGTTTTCAAG TGAAAGCTCT AGATCCCTCC CGAGAAAAAT GAAGCGTAGT CAAAGCGGTA 1560CATATAATTT CAGGGAAGAT GGGGGTCTTC CTAGGTCAGT CATGGACCCC AAGTGAAGTA 1620AGAATTCCTG TTCTAGACTT CCTATTTTCT TTGCAATTTG GATCCTTGAC CAGGGAAGCG 1680AATAAGATTG TATATGAGAT TTAGAGGTTC AGTGAGAATG GTGGCATGAA TACAGAAGAT 1740GTGGTGTTTT TCTGTATCCT TGGCCAGGTG AAGCATCAAG GTCTGAATGA GAACATCAAC 1800CTGGACTCTG TGGATGGTGT GCCAATGGCA AGCACTGATC GATGGAGTGA GCTGACGGAG 1860GCAGAGCGAC TCCAAGAGAA CCTCCGAGCT TACCGTACCT TCCATGTTAT GTTGGCCAGG 1920CTGTTAGAAG ACCAGCGGGA ACATTTTACT CCAGCTGAAG ATGACTTCCA TCAAGCAATA 1980CACACCATTG TCCTCCAAGT CGCTGCCTTT GCTTACCAGC TGGAAGAATT AATGGTGCTC 2040CTGGAGCACA AGGTCCCCCC CAGTGAGGCT GATGGTACGC CCCTCAGCGT TGGAGGTGGT 2100GGTCTCTTTG AGAAGAAGCT GTGGGGCCTG AAGGTGCTGC AAGAGCTTTC ACAGTGGACA 2160GTGAGGTCCA TCCGTGACCT TCGAGTCATC TCCTCTCATC AGGCTGGGGT CCCAGCACAC 2220GGGAGCCATC ATGTCGCTAA GGACAAGAAA ATGTAGCAGT TACCTCCCTT CTTTCTTAGT 2280TGCCTTCTAT TCTAATGGAA TAGACAGTTC TCTGAGGCCT CACTTCCCAT TCTTATTTTT 2340GAAAAAAAGA CTGCAAGCAT TTTTGTAACT AGGGTTGGAG ACATGGACAA ATGGGCATGC 2400AGGTTTAGTG TGAGAGTGTG TGTGCGTTGG GGCCATGAGA GAGCGAGGGC AGGGACGCCC 2460CCACAGTGCA CTAACCTCTC CCTACCCACT AAATACCCTT TACAGACATT TAACAGCCGC 2520ACAGGATAAA TATATTTTTA ACTCTAGTTC TGGATGACTC GTCTGAGAAG ACTTAAATAG 2580TGAATTAAAA ATCACAGAGT CTAGCCAGTT CAAACCCTTG GACAATAAAA ATAGTAACTA 2640AACATTTATT GAGTATCTAC TATATTGAAG CACTATGCCA 2680

(2) sequence number: 51 data:

(i) sequence signature:

(A) length: 4267 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linear

( xi ) ：SEQ ID NO：51：CTCGAGAGGG AGTGAGGGTT AAAACTCTGT GGTGCAACGG AAACGAATCC AACTGGGAAA 60CCATGAGGCT GTGGGTTGGA TCCCCGGCCT CGCTCAATGG GTTAAGGATC CAGCACGGCG 120CTGCCGTGAG CTGTGGTGTA GGTCGCAGAC GAAGCTTGGA TCCCACTTGG CTGTGGCTGT 180GGCTGTGGCT GTGGTGTAGG CCCGCAGCTG TAACTGTAAT TCGACCCCTA GCCTGGGAAC 240CTCCACAAGC CACGGGTGTG GCCCTAAAAA GCAAAAAAAC GAAAGCAAAA AGAACACTCT 300CAAAGCCTAA ACTTTGAGCA AAAAGAACAC TCTCAAAGCC TAAACTTTGA GCAGATGCCT 360TACACCGCCC CCACGCCTCT CATCCCCTTT CTGTCTGGGC CTCCAGCTCC CTTCCCCCTT 420AACCCAGAAA TCCCAGACCT CAGACCCAAG GATTTCGAAT CCCCAGGCCT TGGCCCAATT 480CTATCATCCC AGCACAGGAC AAGAAAAAAG CAGGGCCGGG CCTTCTGGTC CTGCTCCTCT 540CCCTGCCAGC CCACCCCACC AGTGGCATGG AAAAAGCTCC GGAATTACTG GGTGAAAAAA 600ACCTCTTCCA TGGGGGCTGG AATTAGGGGG GGGGTGATGG TTGCCAACCC CACCCCTCCC 660CTCCCTCCCT TCCCCCACCC TGCTGTGTGA AAGGGGAGGC CAGCCCACTT CGTGACCCGA 720CGGGGGCTGG CCCAGCTGGC CCCAGTTCTG GAGGAGTGGG CGGGGCGGGG GGAGCCCTAT 780AATTGGCCGA ATCTGGGCTC CCTGAATCAT ACTCAGCCCC GGAGGAGGAA GGAGGAAGGA 840GGAGGAGGAA GCAACCGGTG AGGAGCAGAC CTGGGGGCAC AGAGATGGGC TCGGGGCTTT 900CGGTGGGGGG GGTGGGCTGT CGGGGGAGGA GGAAATGACC TGGCCCCCCG GGGCCACCAC 960CGAGGCAGGA GTTGGGGATG AGGCTAGAGC CCAGGGACTG GACCTAGAAG GAGGGTGGGC 1020AGCAGGAGGA GGTTATCCGC CTTGGCTGGA AGGGGAGGTC AGGGAAGCAG CGGGACCTGT 1080AGGAAGAACC AGACGAGCCA GAGCCGACGA ATTGTACTGG CAGGTATGGC GCATCTACTC 1140AAGTTTTGAG CACACTAAGA GCTCCATCGA GGAGACCCAG GGGTGGCGGC GACCAGGGGT 1200GACCTCGACC GGGCTGGCGG CAGGGTAGCT AGAGCGTTGG TGGAAGGACA TGTAAATGAG 1260GATTAAATTA GGGAATGAGT GGAAAACAGG GTTTAGATGT GAAGTTGGAG CTTGGAATGT 1320GAAGGTACCA GGAAGAACGT GAGCTTGGAG CCCAGAAAGC AAGGCTGGGG CTCACATGGG 1380ACTCCAGGGT GGAAGGGGTG GGGGGCGACG TGGGTGGAAT TTGAACCCTG GGAAAAAAGG 1440AAGGCTTTTG GCCGCACCCG ACCTGGGGAT GGGGAGATAG GAGAAGACAA TGAGGGAATT 1500ACACGGACAA TGGAAAGGAT CTGCTCGGGA AATATCTGCT TGGATTAGGC TGATGCAGAT 1560AAGGGGGTGC AAGGCTTGGA AGGCTGTGAC TGGACAGGGC TGGGCTCTGG GTGAGAGGAG 1620CGAGCCCCGC CGCTGTTGAG TGACAATTTC TCCCTCCTGC AGGTTGGCCA ATCGCAAGCC 1680AGAAGATGAG GGTTCTGTGG GTTGCTTTGG TGGTAACCCT CCTCGCAGGT ATGGGGGTGG 1740GGCTTGCTCA GGTTCCCTGC CCCTCCCCCA TCCCCGGTGC CCCTCCTTCA TCCCTGGGTC 1800TCTTCTGCTG GTCTCTCTTC CCCTTGAGGA GAGGCCTAGA TGTGAGGCCT CTCTGGCACT 1860CCTTGCTTCT GAACAGCTCG TTTTACTCTC TGAGCCTCAG TTTCCCCATC TTTAAAATGG 1920GAGTTATGTT GAGAGATTCC AGCTGTGGCT CAGCAGGTTA AGAACCCGAC TAGTATCCAT 1980GAGGAAGAGG GTTCAATCCC CTGGCTTCGC TCAGCGGGTT AAGGATCCGG CGTTGCCATG 2040AGCTGCGGCA TAAGTCGCAG ATGCAGCTCG AATCGGGTGT TGCTGTGGCT GTGGTGCAGG 2100CTGGCAGCTA TCGCTTCCAT CGGACCCCTC GCCTGGGAAC TTCCACGTAT GCCACTGGTG 2160CAGCCCTAAA AGACAAACAA ACAAAAACGA AAGAAAGAGA AAAGAAAGGA AAGGGGGCTT 2220CTGTTTCTAA TGCGTTGTTG CCTGGCAGGG CGTGAGCATT AGATACGTGT CAGCTGTGAC 2280TAGCGTGCAC GGAGCACACA ATCCATGCTT GTCCAGTAAT TAGACAGGCT GGGTGTCCTT 2340CCACCCCCTC CCTGCCCACC AGTGCTCTAG AGAAGCCCAC CCACCAGGGC TGGGGGAGCA 2400CCTGCTCTGT ACCAGGTACC GTGTGCTGGG AGGGGGCAGA GGACCTGATG GCTGTGAACT 2460GGCTCGGTGC AGGATGCCGG ACAGAGGACG AGCCGGGGCC GCCGCCGGAG GTGCACGTGT 2520GGTGGGAGGA GCCCAAGTGG CAGGGCAGCC AGCCCTGGGA GCAGGCCCTG GGCCGCTTCT 2580GGGATTACCT GCGCTGGGTG CAGTCCCTGT CTGACCAAGT GCAGGAGGAG CTGCTCAGCA 2640CCAAGGTCAC CCAGGAACTG ACGTAAGTGC CCACCCGACT CCCGCCGCGC GCGCGCGCGC 2700GCGCGCGCGC GCCTGACCCT CCTGGCGAAC CGTGTGTTCT GGACCCTCAG GCTCCACCCG 2760TCCGGGTTTC CTTCTGTCCT TGTCGCCAAC TCTTGGGGGT CTGGGTCTCT GTTTCTTTTT 2820TTTCCTTCCT CCTTTTTTGG GGGGAAAAAA CTTTTTCTTT TTTCTTTCAT TTGACTTCAT 2880GTCTTGCTTT CTTTCCATCT TGAGCTCCTG CCTTCGCCTG TCTCTGGGTC AGTCTTGCCG 2940TCCCTTGCTG TCTCTGAATC TCTGGCACGT CCTGGCCATC GCCAGCTCAG GAGCCCTCCT 3000TCTCCCCCTC ACCGCCCCCG CCCTCTCTGC GCCCAGGGAG CTGATAGAGG AGAGCATGAA 3060GGAGGTGAAG GCCTACCGCG AGGAGCTGGA GGCGCAGCTG GGCCCCGTGA CCCAGGAGAC 3120GCAGGCGCGC CTGTCCAAGG AGCTGCAGGC GGCGCAGGCC CGCGTGGGCG CCGACATGGA 3180GGACGTGCGC AACCGCTTGG TGCTCTACCG CAGCGAGGTG CACAACATGT TGGGCCAGAC 3240CACCGAGGAG CTGCGGAGCC GCCTGGCTTC CCACCTGCGC AAGCTGCGCA AGCGGCTGCT 3300CCGCGACACC GAGGACCTGC AGAAGCGCCT GGCCGTGTAC CAGGCGGGGC TGCGCGAGGG 3360CGCCGAGCGC AGCGTGAGCG CCCTCCGCGA GCGCCTCGGG CCCCTGGTGG AGCAGGGCCG 3420ATTGCGCGCC GCCACCCTGA GTACCAGGGC CGGCCAGCCG CTGCGCGAGC GCGCGGAAGC 3480CTGGGGCCAG AAGCTGCGCG GACGGCTGGA GGAGATGGGC AGCCGGACCC GCGACCGCCT 3540GGATGAGATG CGTGAGCAGC TGGAGGAGGT GCGCACCAAA GTGGAGGAGC AGGGCAGCCA 3600GTTGCGCCTG CAGGCCGAGG GATTCCACGC CCTCCTCAAA GGCTGGTTCG AGCCTCTGGT 3660GGAAGACATA CGGCGCCAGT GGGCCGGGCT GGTGGAGAGG ATGCAGTCGG CCGTGAGCAT 3720AAGCTCCTCC ACCTCTGCGC CCAGTGATAA TCAGTGAGTG CCCTCTCATC CGGGCACCCC 3780CTTCGGGGCC CCGTTCCTGC CCAACTCCCC CGCCTCCCCC AGCCTTAGAT GCCCTCTTGG 3840TGGGCCCCTG CTTAATAAAG ATTCATCAAG CTTCACAGCA GCTTCTGGGT GTCCCCGGTG 3900TGATTTCTCA GCTCCAGCCT CAGTTTCCCT TTCCTTCCCT GCACTGACCA CCCAGTTCTC 3960TGTCCTGCCC TCTGCCTGTG TGTGTCTATT TGTCTCTTCT CCCCCTTTTC TTTTTTTTTG 4020GCCGAGCCCA TGGCATGCGG AAGTTCCCCC GGCCAGGGAT TGAACCCATG CCACAGCCGC 4080CACAACGAAG GATCCTTAAC TACTAGGCCA CCAGGGAACT CCATCCTTTC TAACTCTGTC 4140TTTGCTTTCC CTTTTTTAGC GTTTTAGGGC TGCACCCTCA GCATGTGGAA GTCCCCAGGC 4200TAGGGGTCAA ATTGGCGCTA CAGCTGCCAG CCTACACCAC AGCCCCAGCA ACGCAGGATT 4260CCTCGAG 4267

Claims

1. cultivate the genitaloid method of non-rodent species, comprise that genitaloid a kind of composition that plating contains non-rodent species is about 1.5 * 10 in density ⁵Cells/square cm and about 10 ⁶On the feeder cell between the cells/square cm, comprise the Prostatropin of significant quantity in its substratum.

2. the process of claim 1 wherein that described feeder cell density is about 2 * 10 ⁵Cells/square cm and about 5 * 10 ⁵Between the cells/square cm.

3. claim 1 or 2 method, wherein said feeder cell right and wrong S1/S14 cell or S1-m220 cell.

4. the method for aforementioned arbitrary claim, wherein said feeder cell are STO feeder cell.

5. the method for aforementioned arbitrary claim, wherein said substratum do not comprise the solubility STEM CELL FACTOR that external source adds.

6. the method for aforementioned arbitrary claim, wherein said substratum do not comprise the leukaemia inhibitory factor that external source adds.

7. the method for aforementioned arbitrary claim, wherein said substratum do not comprise the solubility STEM CELL FACTOR that external source adds and do not comprise the leukaemia inhibitory factor that external source adds.

8. the method for aforementioned arbitrary claim, wherein said substratum comprises the Prostatropin of concentration between about 5 nanograms/milliliter and about 100 mcg/ml.

9. the method for aforementioned arbitrary claim, wherein said substratum further comprises the uterus ferritin of significant quantity.

10. the method for aforementioned arbitrary claim, wherein said substratum further comprises the uterus ferritin of concentration between about 1 nanograms/milliliter and about 100 mcg/ml.

11. the method for aforementioned arbitrary claim, wherein said substratum further comprises the alpha2-macroglobulin of significant quantity.

12. the method for aforementioned arbitrary claim, wherein said substratum further comprise the alpha2-macroglobulin of concentration between about 10 nanograms/milliliter and about 10 mcg/ml.

13. what the method for aforementioned arbitrary claim, wherein said substratum further comprised significant quantity is non-essential amino acid to described non-rodent.

14. it is non-essential amino acid to described non-rodent between about 10nM and about 250nM that the method for aforementioned arbitrary claim, wherein said substratum further comprise concentration.

15. the method for aforementioned arbitrary claim, wherein said substratum further comprise the L glutamine of significant quantity.

16. the method for aforementioned arbitrary claim, wherein said substratum further comprise the L-glutaminate of concentration between about 0.1mM and about 50mM.

17. the method for aforementioned arbitrary claim, wherein said substratum further comprises the beta-mercaptoethanol of significant quantity.

18. the method for aforementioned arbitrary claim, wherein said substratum further comprise the beta-mercaptoethanol of concentration between about 1 μ M and about 1mM.

19. the method for aforementioned arbitrary claim, wherein said substratum comprises the Prostatropin of significant quantity and in conjunction with the material below at least two kinds of significant quantity: the uterus ferritin, alpha2-macroglobulin, to described non-rodent nonessential amino acid, L-glutaminate or beta-mercaptoethanol.

20. the method for aforementioned arbitrary claim, wherein said substratum comprise the improved Eagle substratum of Dulbecco, the mixture of improved Eagle substratum of the Dulbecco of Ham ' s F10 substratum or 50: 50 volume/volume and Ham ' s F10 substratum.

21. the process of claim 1 wherein that described substratum further comprises the leukaemia inhibitory factor of significant quantity.

22. the method for claim 21, wherein said substratum advance to comprise the leukaemia inhibitory factor of concentration between about 5 nanograms/milliliter and about 100 mcg/ml.

23. the process of claim 1 wherein that described substratum further comprises the solubility STEM CELL FACTOR of significant quantity.

24. the method for claim 23, wherein said substratum comprise the solubility STEM CELL FACTOR of concentration between about 1 nanograms/milliliter and about 100 mcg/ml.

25. the process of claim 1 wherein that described substratum comprises the Prostatropin of significant quantity and in conjunction with uterus ferritin, alpha2-macroglobulin and the leukaemia inhibitory factor of significant quantity.

26. the method for claim 25, wherein said substratum comprises the Prostatropin of concentration between about 5 nanograms/milliliter and about 100 mcg/ml, the uterus ferritin of concentration between about 1 nanograms/milliliter and about 100 mcg/ml, concentration be the leukaemia inhibitory factor between about 5 nanograms/milliliter and about 100 mcg/ml at alpha2 Macroglobulin between about 10 nanograms/milliliter and about 10 mcg/ml and concentration.

27. the process of claim 1 wherein that described substratum comprises:

(a) Prostatropin between about 5 nanograms/milliliter and about 100 mcg/ml;

(b) the uterus ferritin between about 1 nanograms/milliliter and about 100 mcg/ml;

(c) alpha2-macroglobulin between about 10 nanograms/milliliter and about 10 mcg/ml;

(d) leukaemia inhibitory factor between about 5 nanograms/milliliter and about 100 mcg/ml;

(e) the solubility STEM CELL FACTOR between about 1 nanograms/milliliter and about 100 mcg/ml;

(f) non-essential amino acid between about 10nM and the about 250nM;

(g) L-glutaminate between about 0.1mM and the about 50mM;

(h) beta-mercaptoethanol between about 1 μ M and the about 1mM;

(i) the improved Eagle substratum of the Dulbecco of about 50% volume/volume; With

(j) Ham ' the s F10 substratum of about 50% volume/volume.

28. the method for aforementioned arbitrary claim, wherein the primordial germ cells of plating were maintained between about 2 generations of undifferentiated state and about 14 generations.

29. the method for aforementioned arbitrary claim, the primordial germ cells of wherein said plating are cultivated for some time, can effectively provide the primordial germ cells derived cell of described non-rodent species to be in the period at this section.

30. the method for aforementioned arbitrary claim, wherein said primordial germ cells comprise at least one first exogenous dna fragment.

31. the method for aforementioned arbitrary claim wherein saidly comprises genitaloid composition and is provided to selected DNA section, and the primordial germ cells that contain selected DNA section are selected.

32. the method for aforementioned arbitrary claim, wherein said primordial germ cells are by electroporation, and particle bombardment method or viral conversion method are provided at least a first exogenous dna fragment.

33. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided to the selected proteinic first dna encoding district of at least a coding.

34. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided to the proteinic first dna encoding district of at least a coding selection marquee.

35. the method for aforementioned arbitrary claim, wherein said primordial germ cells be provided at least a in described non-rodent species cell the first dna encoding district of expressing green fluorescent protein matter.

36. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided at least a coding and list in the proteinic first dna encoding district in table 3 or the table 4.

37. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided at least a first and a kind of second dna encoding district, their at least a first and a kind of second selected protein of encoding.

38. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided to a kind of selected proteinic first dna encoding district of at least a coding and the proteinic second dna encoding district of at least a coding selection marquee.

39. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided at least a first dna encoding district, it effectively is positioned under the promotor control of expressing described dna encoding district in described primordial germ cells.

40. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided at least a first dna encoding district, it effectively is placed on CMV, and under Oct-4 or the control of pgk promotor, described promotor is expressed described dna encoding district in described primordial germ cells.

41. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided at least a first dna encoding district, it is effectively to be placed on a promotor control in the other direction down, and this promotor is the antisence product in described primordial germ cells.

42. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided to contain two DNA sections that are positioned at the selected DNA district of its flank, instruct described DNA section homologous recombination thus in the genomic dna of described non-rodent species.

43. the method for claim 42, wherein said selected DNA district comprises the genomic dna from described non-rodent species.

44. the method for claim 43, wherein said selected DNA district comprises the genomic dna from the Oct-4 gene.

45. the method for aforementioned arbitrary claim, wherein said primordial germ cells are provided to contain two DNA sections that are positioned at the selected dna sequence dna of its flank, instruct the excision of described DNA section under the condition of being fit to thus.

46. the method for claim 45, wherein said selected dna sequence dna is the loxP site.

47. the method for aforementioned arbitrary claim, wherein said primordial germ cells are oxen, sheep, pig or caprine primordial germ cells.

48. the method for aforementioned arbitrary claim, wherein said primordial germ cells are pig primordial germ cells.

49. the primordial germ cells of the non-rodent that the method by aforementioned arbitrary claim produces.

50. the primordial germ cells derived cell system of the non-rodent that produces by method one of any among the claim 29-48.

51. produce the method for the non-rodent of transgenosis, comprising:

(a) contain genitaloid composition from described non-rodent embryo separation;

(b) introduce selected DNA section and enter the genitaloid composition that contains described non-rodent contains selected DNA section with acquisition candidate's primordial germ cells;

(c) plating contain selected DNA section described candidate's primordial germ cells on feeder cell, described feeder cell density is about 10 ⁵Cells/square cm and about 10 ⁶Between the cells/square cm, in substratum, contain the Prostatropin of significant quantity, contain the primordial germ cells of the described non-rodent of selected DNA section with acquisition; With

(d) primordial germ cells from the non-rodent that contains selected DNA section produce the non-rodent of transgenosis, and wherein selected DNA section contains in the somatocyte of described non-rodent or sexual cell and expresses.

52. the method for claim 51 wherein saidly comprises genitaloid composition and contains culturing cell from primordial germ cells derived cell system.

53. the method for claim 51 or 52, the method that wherein produces the non-rodent of described transgenosis comprises that the primordial germ cells of injecting the described non-rodent species that contain selected DNA section enter the blastocyst of described non-rodent species.

The method that one of 54. claim 51-53 is any, the method that wherein produces the non-rodent of described transgenosis comprises:

(a) inject the primordial germ cells of the described non-rodent species that contain selected DNA section in blastocyst from described non-rodent species;

(b) shift the non-rodent that described blastocyst is become pregnant with generation in the synchronization female receptor of described non-rodent species; With

(c) make the gestation in the described non-rodent of becoming pregnant be enough to allow the time of growing for the non-rodent of transgenosis that survives.

55. the method for claim 51 or 52, the production method of the non-rodent of wherein said transgenosis comprise from the described enucleation oocyte of examining described non-rodent species of the primordial germ cells separating nucleus of the described non-rodent species that contain selected DNA section and injection.

The method that one of 56. claim 51,52 or 55 is any, the production method of the non-rodent of wherein said transgenosis comprises:

(a) from the described enucleation oocyte of examining described non-rodent species of the primordial germ cells separating nucleus of the described non-rodent species that contain selected DNA section and injection;

(b) shift the non-rodent that described ovocyte is become pregnant with generation in the synchronization female receptor of described non-rodent species; With

57. the method for claim 51 or 52, the method that wherein produces the non-rodent of described transgenosis comprise that the early embryo of the primordial germ cells that make the described non-rodent species that contain selected DNA section and described non-rodent species assembles.

The method that one of 58. claim 51,52 or 57 is any, the production method of the non-rodent of wherein said transgenosis comprises:

(a) primordial germ cells of the described non-rodent species that contain selected DNA section and the early embryo of described non-rodent species are assembled;

(b) shift the non-rodent that described embryo is become pregnant with generation in the synchronization female receptor of described non-rodent species; With

The method that one of 59. claim 51-58 is any, the non-rodent of wherein said transgenosis is an ox, sheep, pig or billy goat.

The method that one of 60. claim 51-59 is any, the non-rodent of wherein said transgenosis is a pig.

61. the non-rodent of transgenosis by one of any method preparation of claim 51-60.

62. a composition comprises:

(a) from a kind of primordial germ cells of non-rodent species;

(b) be enough to reach about 1.5 * 10 ⁵With 10 ⁶The feeder cell of density between feeder cell/square centimeter; With

(c) Prostatropin that exists with described genitaloid growth of effective promotion and continuous amount of breeding.

63. the composition of claim 62, wherein said primordial germ cells comprise at least a first foreign DNA section.

64. the composition of claim 62 or 63, wherein said feeder cell are STO cells.

The composition that one of 65. claim 62-64 is any, one or more the following material that further comprises the significant quantity that promotes described primordial germ cells growth and breed continuously, the uterus ferritin, alpha2-macroglobulin, leukaemia inhibitory factor, solubility STEM CELL FACTOR, to described non-rodent species are non-essential amino acids, L-glutaminate, beta-mercaptoethanol, the improved Eagle substratum of Dulbecco or Ham ' s F10 substratum.

The composition that one of 66. claim 62-65 is any, wherein said primordial germ cells are oxen, sheep, pig or caprine primordial germ cells.

The composition that one of 67. claim 62-66 is any, wherein said primordial germ cells are pig primordial germ cells.

The purposes of the composition that one of 68. claim 62 to 67 is any in a kind of primordial germ cells derived cell of preparation system.

69. be used to prepare one of any composition of the claim 62 to 67 of a kind of primordial germ cells derived cell system.

The purposes of the composition that one of 70. claim 63 to 67 is any in the non-rodent of a kind of transgenosis of preparation.

71. be used to prepare one of any composition of the claim 63 to 67 of the non-rodent of transgenosis.

72. a test kit is comprising one of any a kind of composition of the claim 62-67 that packs with appropriate containers.