CN100526460C

CN100526460C - Transgenic ungulates having reduced prion protein activity and uses thereof

Info

Publication number: CN100526460C
Application number: CNB2003801084165A
Authority: CN
Inventors: J·罗布尔; Y·库罗伊瓦; K·托米祖卡; I·伊施达
Original assignee: Kirin Pharma KK
Current assignee: Kyowa Kirin Co Ltd
Priority date: 2002-11-08
Filing date: 2003-11-10
Publication date: 2009-08-12
Anticipated expiration: 2023-11-10
Also published as: CN1742088A

Abstract

The invention provides cloned transgenic ungulates (e.g., bovines) in which prion protein activity is reduced by one or more genetically engineered mutations. Desirably, these transgenic bovines are also genetically modified to express xenogenous (e.g., human) antibodies. Because of their resistance to prion-related diseases such as bovine spongiform encephalopy (also known as mad cow disease), these bovines are a safer source of human antibodies for pharmaceutical uses and safer source of agricultural products.

Description

Transgenic ungulates that prion protein activity reduces and uses thereof

Technical background

In general, the invention is characterized in clone's transgenic ungulates (for example ox), wherein the activity of prion protein (PrP) is lowered by one or more genetically engineered sudden changes.Since the active transgenic cattle that reduces of this Protein virus to the Protein virus relative disease for example mad cow disease (BSE is also referred to as mad cow disease) have resistance, so they are safer and preferred sources of agricultural-food and medicine such as people's therapeutic antibodies.

Since Britain in 1986 found the first BSE, this communicable disease had propagated into other areas in the world, for example Japan.The harm of this disease produces influence greatly to agricultural and pharmacy industry, has limited the use of dairy products in these industries.

Based on research during the last ten years, it is the real inducement of this communicable disease that Protein virus has been determined.The animal that prion protein activity reduces has the resistance to the prion protein infections relating of expectation, therefore, is that people expect.

So far, do the gene targeting method of using routine in (ES) cell, created the transgenic mice that prion protein activity reduces the mouse embryo who carries out homologous recombination easily.But for example the ES cell of ox separates, cultivation and genetic modification are difficult to ungulate.

Because the expression of prion protein gene in fetal fibroblast is very active; in the generation sheep that prion protein knocks out, with promoterless knocking out (KO) carrier transfection fetal fibroblast (Denning etc., Nature Biotech.; 19:559-562,2001).By using this type of carrier, because the promoterless drug resistance gene (for example anti-Xin Meisu or anti-tetracycline gene) in the knockout carrier is only expressed when time on the locus that is integrated into active expression, therefore, can use for example G418 or the tetracycline clone that selects homology to practice shooting of suitable medicine.

Yet the sheep inoblast of practicing shooting by these hemizygotes of nuclear transplantation only obtains a lamb alives, and the just death in about 12 days after birth of this lamb.Unlimited long-life mouse ES cell is different with having, and fibroblastic life-span of body is limited and since carry out that strict drug screening selects to expect knock out cell the time need the time of culturing cell long, make that the somatic cell gene target practice is very difficult.In addition, generally speaking, the frequency ratio mouse ES cell that homologous recombination takes place in the body inoblast will hang down about 10-100 doubly.The domestic animal alive that the low success rate of the nuclear transplantation method of restriction that these are relevant with the body inoblast and routine makes preparation have the some specific mutant of expectation is difficult.

In ox, carried out reducing the trial of prion protein activity by gene targeting.As far as we know, also report is not successfully produced the transgenic cattle that has sudden change on the prion gene seat, and this may be owing to lack suitable knockout carrier and/or nuclear transplantation method.Therefore, need suddenly change effectively prion gene seat in the ungulate cell (for example ox cell) of improved knockout carrier.In addition, being used for producing improving one's methods of transgenic ungulates (for example ox) from the donorcells of these genetic modifications also expects.

Summary of the invention

The invention is characterized in the design knockout carrier, use this carrier, can for example on the prion gene seat of ungulate (for example ox), realize high-frequency hemizygote and homozygote target practice integration (so-called homologous recombination) in the fetus body inoblast at donorcells.Feature of the present invention also is to have on the prion gene seat with the donor preparation of genetic modification the calf of the work of hemizygote or homozygote sudden change in any nuclear transplantation method described herein.These oxen can be used to prepare medicine and agricultural-food, for example therapeutic people's antibody of human.

Method of the present invention is to adopt multiple technologies, for example (i) prion gene described here knocks out cell, (ii) the karyomit(e) of describing among the open W002/051997 of cloning of mammalian animal method such as nuclear transplantation or PCT transplant and (iii) with artificial chromosome (HAC) for example δ HAC import ungulate (the open W002/70648 of PCT; Kuroiwa etc., Nature Biotechnol.20:889-894,2002) realize.In the cloning of mammalian animal method, the ungulates cell that Protein virus knocks out is used as the source of donor genetic material, produces the offspring that Protein virus knocks out (hemizygote or homozygote).

The ungulate that Protein virus knocks out also has other useful feature, for example produces people's antibody.Can adopt the combination of above-mentioned technology to produce this ungulate.For example, can by the hybridization ungulate that knocks out of Protein virus and as the PCT ungulate that discloses the production people antibody of describing among the W002/70648 generate that Protein virus knocks out and produce the ox of people's antibody.No matter whether carry out breeding of ungulate, the operate continuously fetal fibroblast also can be used to produce this ungulate.The operate continuously of bovine fetal fibroblast comprises and is repeated below step: (i) inoblast of genetic manipulation ungulate (for example ox), (ii) use this cell to carry out cloning of mammalian animal, (iii) fetus generates, with the fetal fibroblast that (iv) separates genetic modification.For example, can operate the inoblast that Protein virus knocks out continuously and keep HAC, and deactivation endogenous Ig gene.

The mono-clonal or the polyclone heteroantibody that generate have various uses; For example, be used as for example composition of the composition of bacterium or virus infection of prevention or treatment pathogenic micro-organism.

Transgenic ungulates and ungulate cell

An aspect, the invention provides a kind of ungulate (for example ox) or ungulate cell (for example ox cell), it has the sudden change (sudden change behind for example initial ATC codon, for example sudden change in 10,20,50 or 100 Nucleotide of this codon) that non-natural takes place on one or two allelotrope of endogenous Protein virus nucleic acid.Preferably, this sudden change reduces or eliminates basically the expression of functional prion protein.In a preferred embodiment, expression functional or whole prion proteins have reduced at least 10,20,40,60,80,90,95 or 100%.Sudden change is a hemizygote or homozygous.In some embodiment, sudden change comprises positive selectable marker (for example antibiotics resistance gene) is inserted in the Protein virus nucleic acid.Preferably, positive selectable marker is operably connected on the xenogeneic promotor.Ungulate or ungulate cell for insert antibiotics resistance gene on two allelotrope of Protein virus nucleic acid may contain identical or different antibiotics resistance gene on each allelotrope.In a preferred embodiment, negative selection marker (for example DT-A or Tk) is operably connected on the xenogeneic promotor, and is present on the allelic carrier of endogenous Protein virus that is used for suddenling change.This sudden change may comprise or not comprise the deletion of the one or more Nucleotide (for example successive Nucleotide) in the Protein virus nucleic acid.

In the preferred embodiment aspect above-mentioned, ungulate (for example ox) or ungulate cell (for example ox cell) have one or more transgenosiss, and express mRNA or by the protein (for example antibody) of transgenes encoding.Preferred ungulate contains the arrangement section (for example human chromosome fragment) naturally of human chromosome or comprises the artificial chromosome of the human chromosome fragment (that is, with respect to the people's gene group, this fragment is rearranged) of artificial through engineering approaches.In some embodiments, heterologous nucleic acid is included in the chromosome segment.This nucleic acid is integrated in the karyomit(e) of ungulate or keeps being independent of host chromosome in the ungulate cell.In various embodiments, nucleic acid is comprised among chromosome segment such as Δ HAC or the Δ Δ HAC.In other embodiments, heteroantibody is the antibody from other kinds, for example people's antibody.

Preferred ungulate and ungulate cell contain one or more heteroantibody locus (at least a xenogenesis Ig molecule takes place to reset and express for all or part of nucleic acid of the heteroimmunity of for example encoding sphaeroprotein (Ig) gene, this immunoglobulin gene) that have in one or more B cells.Preferably, nucleic acid has the light chain nucleic acid fragment of the antibody that is not rearranged, and wherein separates with the nucleic acid fragment of coding J gene fragment by will encode all nucleic acid fragments of V gene fragment of one or more Nucleotide.Other preferred nucleic acid have the heavy chain of antibody nucleic acid fragment that is not rearranged, and wherein (i) separates with the nucleic acid fragment of all encoding D gene fragments and/or (ii) separate with the nucleic acid fragment of all coding J gene fragments by the nucleic acid fragment of one or more Nucleotide with all encoding D gene fragments by the nucleic acid fragment of one or more Nucleotide with all coding V gene fragments.Other preferred ungulates have all or part of nucleic acid of heteroimmunity sphaeroprotein (Ig) gene that one or more coding resets, this immunoglobulin gene at least a xenogenesis Ig molecule of encoding.

In other embodiment preferred, the light chain of heteroantibody and/or heavy chain are by people's nucleic acid encoding.In preferred embodiments, heavy chain is the heavy chain of any kind, for example μ, γ, δ, ε or α, and light chain is λ or K light chain.In other embodiment preferred, the nucleic acid of coding heteroimmunity globulin chain or antibody is the form that is not rearranged.In other embodiment preferred, generate more than one heteroantibodies by ungulate.In various embodiments, generate more than one different xenogenesis Ig or antibody by ungulate.Heteroantibody can be mono-clonal or polyclonal antibody.

In the various embodiments aspect above-mentioned, ungulate (for example ox) or ungulate cell (for example ox cell) have the sudden change that reduces the heteroantibody expression.Preferably, this sudden change reduces the expression of functional IgM heavy chain or eliminates the expression of functional IgM heavy chain basically.In other embodiment preferred, this sudden change reduces the expression of functional Ig light chain or eliminates the expression of functional Ig light chain basically.In other preferred embodiments that also have, this sudden change reduces the expression of functional IgM heavy chain and functional Ig light chain, perhaps eliminates the expression of functional IgM heavy chain and functional Ig light chain basically.Preferably, ungulate has sudden change on one or two allelotrope of the endogenous nucleic acid of coding β-(l, 3)-galactoside transferase and/or J chain.In other preferred embodiments, ungulate has the nucleic acid of coding xenogenesis J chain such as people's J chain.Preferably, this sudden change reduces or eliminates the expression of endogenous β-(l, 3)-galactoside transferase, galactosyl (α l, 3) semi-lactosi epi-position and/or J chain.Preferably, ungulate produces people's IgA or contains the IgM molecule of people J chain.Preferred ungulate cell (for example ox cell) comprises somatocyte, for example fetal fibroblast or B cell.

Feature of the present invention also is to produce the hybridoma of xenogenesis (for example people) antibody.In one aspect, the invention provides by B cell of the present invention and myeloma cell and merge the hybridoma that forms.Preferably, antibody and interested antigen have reactive behavior.

Produce the method for transgenic ungulates cell

Feature of the present invention also is to produce the method for the ungulate cell (for example ox cell) that has sudden change on one or two allelotrope of Protein virus nucleic acid.These cells can be used as donorcells and produce genetically modified ungulate (for example bovine prion protein knock out ungulate).Preferably, this sudden change makes the content of functional prion protein reduce and/or infection or the disease for example BSE minimizing relevant with Protein virus.

Therefore, in one aspect in, the invention is characterized in the method for preparing transgenic ungulates (for example ox).This method is included under the condition of first allelotrope generation homologous recombination that allows the endogenous Protein virus nucleic acid in first carrier and the cell, first Protein virus targeting vector is imported in ungulate cell, thereby hemizygote is suddenlyd change in the transfered cell.Preferably, first carrier comprises first homology zone, the positive selectable marker with sequence substantially the same with first zone of the endogenous Protein virus nucleic acid of this cell and has second homology zone with the substantially the same sequence in second zone of the endogenous Protein virus nucleic acid of this cell.In preferred embodiments, a homology zone is at least than another homology zone long 1,2,3,4,5,6 or 8kb.Preferably, this method also is included between second allelotrope that allows first carrier and endogenous Protein virus nucleic acid, in cell, take place under the condition of homologous recombination first carrier to be imported in the cell once more, thereby in cell, import the homozygote sudden change.In other embodiments, this method also is included between second allelotrope that allows second carrier and endogenous Protein virus nucleic acid, in cell, take place under the condition of homologous recombination, to have in second prion gene targeting vector transfered cell of the antibiotics resistance gene that is different from first carrier, thereby in cell, import the homozygote sudden change.Preferably, when having the 1mM spermidine, with in first and/or second the carrier transfered cell.Preferred cell comprises bovine fetal fibroblast.

In various embodiments of the present invention, be used to suddenly change endogenous Protein virus nucleic acid nucleic acid (for example, comprise nucleic acid that is operably connected to the selectable mark of coding and the box that knocks out that selectively is connected to promotor on the nucleic acid with sequence substantially the same with Protein virus nucleic acid) be not comprised in virus vector, for example in adenovirus carrier or the adeno-associated virus vector.For example, can adopt standard method for example not relate to the transfection or the fat transfection of the virus transfection of cell, this nucleic acid is comprised in plasmid or the artificial chromosome, plasmid or artificial chromosome are imported in the ungulate cell.In also having another embodiment, the nucleic acid (for example comprise the nucleic acid that is operably connected to the selectable mark of coding and selectively be connected to the expression cassette that knocks out of promotor on the nucleic acid with sequence substantially the same with Protein virus nucleic acid) of endogenous Protein virus nucleic acid of will being used to suddenly change is included in virus vector, for example in adenovirus carrier or the adeno-associated virus vector.According to this embodiment, the virus that will contain virus vector is used to infect the ungulate cell, makes insertion portion or whole virus vector in the ungulates cell.

The method for preparing transgenic ungulates

The present invention also is provided for preparing the method that has the transgenic ungulates of one or more sudden changes in endogenous Protein virus nucleic acid.This method a kind of comprises any cell of above-mentioned aspect of the present invention, is inserted in the ovocyte from the chromatin of this cell or from the nucleus of this cell.This cell has first sudden change in endogenous Protein virus nucleic acid.Ovocyte or fetal development are become under the condition of fetus, and the embryo transfer that forms with ovocyte or by this ovocyte is in the uterus of host ungulate.Preferably, this fetation offspring of becoming to survive.

In preferred embodiments, the nucleic acid that comprises box by insertion is with in first sudden change transfered cell, this box comprises on the nucleic acid that is operably connected to the selectable mark of coding and is operably connected to one or more promotor that has on the nucleic acid of the sequence substantially the same with endogenous Protein virus nucleic acid that this box is integrated on the endogenous allelotrope of Protein virus nucleic acid.In other embodiment preferred, the nucleic acid that comprises first box by insertion is with in the sudden change transfered cell, but this box comprises on the nucleic acid that is operably connected to first selective marker of coding and is operably connected to first promotor on the nucleic acid that article one has the sequence substantially the same with endogenous Protein virus nucleic acid, thereby first box is incorporated on first endogenous allelotrope of Protein virus nucleic acid, generates first transgenic cell.Insert the nucleic acid that comprises second box in first transgenic cell, but this box comprises on the nucleic acid that is operably connected to second selective marker of coding and is operably connected to second promotor on the nucleic acid that second has the sequence substantially the same with endogenous Protein virus nucleic acid.Second selectable marked differential be in first selectable mark, and second box be integrated on second the endogenous allelotrope of Protein virus nucleic acid, generates second transgenic cell.

In also having other embodiment preferred, isolated cell the offspring who generates from embryo, fetus or by fetus imports to other sudden changes in the Protein virus nucleic acid or another kind of nucleic acid (for example heavy chain of antibody or light chain nucleic acid) of this cell.Then with the cell that generates, from the chromatin of this cell or carry out second from the nucleus of this cell and take turns nuclear transplantation, generate transgenic ungulates with two or more sudden changes.This sudden change occurs on a kind of identical or different allelotrope of gene or occurs on the different genes.Be used for the first round or the second optional cell coding heteroantibody of taking turns nuclear transplantation.In specific embodiment, this cell comprises all or part of nucleic acid of one or more coding xenogenesis Ig gene, and one or more xenogenesis Ig molecule can take place to reset and express in the B cell these Ig genes.In preferred embodiments, the cell that is suddenlyd change is inoblast (coming fetal fibroblast).Preferably, the endogenous gene that is suddenlyd change is operably connected on the endogenous promotor, and this promotor is non-activity in inoblast.In other embodiment preferred, the specific activity that is operably connected to the endogenous promotor on the endogenous gene of sudden change is operably connected to for example activity low 80,70,60,50,40,30,20,10% of the endogenous promotor of GAPDH of endogenous house-keeping gene.Can adopt standard analysis to measure the activity of promotor, for example measure mRNA or by the analysis of the proteinic level of genes encoding (referring to, CurrentProtocols in Molecular Biology such as Ausubel for example, the 2nd volume, the 11.13.1-11.13.3 page or leaf, JohnWiley﹠amp; Sons, 1995).This homotopic method of production transgenosis ungulates that is used for has the advantage that the gene of not expressed at the donorcells cell of the genetic material of nuclear transplantation source (promptly as) is undergone mutation.

Preferably, the cell that is used to produce transgenic ungulates has sudden change on one or two allelotrope of the endogenous nucleic acid of coding β-(l, 3)-galactoside transferase and/or J chain.In other embodiment preferred, this cell has coding xenogenesis J chain, the nucleic acid of people J chain for example, the nucleic acid of the heteroantibody of perhaps encoding.Preferably, heterologous nucleic acid coding xenogenesis Ig gene all or part of, rearrangement and more than one xenogenesis Ig molecule of encoding can take place in this gene in the B cell.In other embodiment preferred, this antibody is polyclonal antibody.In also having other embodiment preferred, immunoglobulin chain or antibody are expressed in serum and/or the milk.

The inventor before disclosed various the improving one's methods of cloning mammal (for example ungulate such as ox) that are used for, be used to be cloned in the Mammals that has one or more sudden changes on the Protein virus nucleic acid (referring to, the open W002/051997 of U.S. Patent Publication 2002-0046722 Al and PCT).In some these methods, cultivate with the substratum of programming again (reprogrammingmedia) (for example cell extract) cell changed processing thoroughly with allow in cell, to add or from cell the various factors of removal, the plasma membrane that seals the cell of being changed processing thoroughly then again is with the factor that comprises expectation and the integrity of recovering cytolemma.In these methods some also comprise donor nuclei (for example separated nucleus in the donorcells) simmer down to chromatin, for example can promote that to discharge the nucleus composition for nuclear transfer embryo is grown be that the offspring of work is the transcription factor of the genetic transcription do not expected.If desired, the step of any of these method can repeat one or many or can sequentially carry out different reprogramming methods to increase the degree of programming again, makes the clone fetus have bigger viability.

A kind of method that is used to produce transgenic ungulates (for example ox) comprises, allowing from nucleus, chromatin or the karyomit(e) of the cell of being changed processing thoroughly, to remove composition (for example nucleus or tenuigenin composition, transcription factor for example) or in nucleus, chromatin or karyomit(e) add under the condition of composition, cultivate the quilt of any above-mentioned aspect of the present invention with the substratum of programming again (for example cell extract) and change the cell (cell that for example in endogenous Protein virus nucleic acid, has one or more sudden changes) of processing thoroughly, thereby generate the cell of programming again.The cell of programming again is inserted in the non-nucleus egg mother cell, and ovocyte or fetal development are become under the condition of fetus, transfer in the uterus of host ungulate with the ovocyte of generation or by the embryo that this ovocyte forms.In preferred embodiments, allowing to form under the chromatinic condition, the cell that to be changed processing thoroughly contacts with following one or more: have or lack anti--mitotic division extract, washing agent and/or salts solution during NuMA antibody, perhaps protein kinase solution.In also having another embodiment preferred, substratum (for example interval cell extract) cultivation of programming again of the cell of being changed processing thoroughly and interval.In also having another embodiment preferred, changed the combination that nucleus in the cell of processing remains film thoroughly, and the karyomit(e) in the nucleus does not concentrate in the culture medium culturing process of programming again with interval.In certain embodiments, in the substratum of programming again, cultivate 50,40,30,20,10 or 5% the dna replication dna of being less than that the cell changed processing thoroughly can not cause dna replication dna or only cause this cell.In other embodiments, in the substratum of programming again, cultivate the cell of being changed processing thoroughly, at least 60,70,80,90,95 or 100% cell, cause dna replication dna.In various embodiments, by with proteolytic enzyme for example Regular Insulin, washing agent for example digoxin or bacteriotoxin for example streptolysin O cultivate intact cell and generate the cell of being changed processing thoroughly.In preferred embodiments, before being inserted into ovocyte, the film of the cell that allows to programme again do not cultivate the cell of programming again under the condition of sealing again.In also having another embodiment, before being inserted into ovocyte, the film of the cell that allows to programme again cultivates the cell of programming again under the condition of sealing again.In other preferred embodiments, the ovocyte of reconstruct or the embryo of generation are with than having equal amts and from horizontal expression nuclear lamina protein A, lamin C or the NuMA albumen of at least 5 times of the expressed respective horizontal height of the contrast ovocyte of same species or contrast embryo.

In yet another aspect, the invention provides the another kind of method that is used to produce transgenic ungulates (for example ox).This method comprises that (a) do not cause the donor nuclei of cultivating under the condition of dna replication dna from cell of the present invention (nucleus that for example has one or more sudden changes in endogenous Protein virus nucleic acid) allowing chromatin to form, (b) chromatin is inserted in the non-nucleus egg mother cell, thereby form the nuclear transplantation ovocyte and (c) be under the condition of fetus, transfer in the uterus of host ungulate with the nuclear transplantation ovocyte or by the embryo that the nuclear transplantation ovocyte forms allowing nuclear transplantation ovocyte or fetal development.In preferred embodiments, allowing in the chromatin of nucleus or generation, to add or removing nucleus or tenuigenin composition for example under the proteic condition of transcription factor, repressor or Chromatin Remodeling, with the substratum of programming again (for example cell extract) cultivation donor nuclei.Preferably, under the condition that allows chromatin to form, the following material of donor nuclei and one or more contact: exist or shortage resists-mitotic division extract, washing agent and/or salts solution during NuMA antibody, perhaps protein kinase solution.The ovocyte of reconstruct or the embryo of generation are with than the cell with equal amts and from horizontal expression nuclear lamina protein A, lamin C or the NuMA albumen of at least 5 times of the expressed respective horizontal height of the contrast ovocyte of same species or contrast embryo.Preferably, this nucleus has the homologous chromosomes that is less than quadruplet (that is, have be less than two pairs of complete chromatids).

Produce the preferred method of chimeric ungulate

Other preferred ungulate is to use the chimeric ungulate from two or more embryos' cell preparation.For example, can with from the cell of nuclear transfer embryo (for example by cell, nucleus or chromatin being inserted into the embryo who forms in the non-nucleus egg mother cell) with combine from cell in vitro fertilization, natural or parthenogenesis activated embryo.Preferably, in the fetal tissue of most of chimeric embryo that is incorporated into generation from the cell and the offspring thereof of nuclear transfer embryo.Cell and offspring thereof from second kind of embryo preferably is attached in the placenta tissue to small part, and strengthens the survival ability of the chimeric embryo that generates.In preferred embodiments, nuclear transfer embryo has sudden change in endogenous Protein virus nucleic acid, and has the nucleic acid of coding heteroantibody.

Therefore, in one aspect, the invention provides by (for example cell of the present invention, nucleus or chromatin, in endogenous Protein virus nucleic acid, have sudden change and selectively have cell, nucleus or the chromatin of nucleic acid of one or more coding heteroantibodies) insert ovocyte and prepare the method for transgenic ungulates, thus generate first kind of embryo.From one or more cells of first kind of embryo and one or more cells contacting, thereby form the third embryo from second kind of embryo.Second kind of embryo or parthenogenesis activated embryo that the embryo is embryo in vitro fertilization, natural generation.Allowing the third fetal development to become under the condition of fetus the third embryo transfer in the uterus of host ungulate.

In another related fields, the invention provides the method that also has another kind of production transgenic ungulates (for example ox).This method is included in permission and removes from nucleus, chromatin or the karyomit(e) of the cell of being changed processing thoroughly under a kind of factor or the condition by the substratum a kind of factor of adding in nucleus, chromatin or the karyomit(e) of programming again, in the substratum of programming again (for example cell extract), cultivate quilt of the present invention and change the cell of processing (for example in endogenous Protein virus nucleic acid, having the cell that suddenlys change and randomly have the nucleic acid of one or more coding heteroantibodies) thoroughly, thereby form the cell of programming again.The cell of will programming again is inserted in the non-nucleus egg mother cell, thereby forms first kind of embryo.From one or more cells of first kind of embryo and from one or more cells contacting in vitro fertilization, natural or second kind of embryo of parthenogenesis activated, generate the third embryo.Allowing the third fetal development to become under the condition of fetus the third embryo transfer in the uterus of host ungulate.

In preferred embodiments, allow nucleus or the tenuigenin composition condition that for example transcription factor is added into or removes from the chromatin of nucleus or generation under, substratum (for example cell extract) is cultivated the cell of being changed processing thoroughly with programming again.In other preferred embodiments, under the condition that allows chromatin to form, the cell of being changed processing is thoroughly contacted: have or lack anti--mitotic division extract, washing agent and/or salts solution during NuMA antibody, perhaps protein kinase solution with following one or more.In also having another preferred embodiment, the cell that will be changed processing thoroughly with interval the substratum (for example interval cell extract) of programming again cultivate.In also having another preferred embodiment, the nucleus of being changed thoroughly in the cell of processing remains membrane-bound, and the karyomit(e) in the nucleus does not concentrate in the culture medium culturing process of programming again with interval.In certain embodiments, in the substratum of programming again, cultivate 50,40,30,20,10 or 5% the dna replication dna of being less than that the cell changed processing thoroughly can not cause dna replication dna or only cause this cell.In other embodiments, in the substratum of programming again, cultivate the cell of being changed processing thoroughly, at least 60,70,80,90,95 or 100% cell, cause dna replication dna.In various embodiments, by with proteolytic enzyme for example Regular Insulin, washing agent for example digoxin or bacteriotoxin for example streptolysin O cultivate intact cell and generate the cell of being changed processing thoroughly.In preferred embodiments, before being inserted into ovocyte, the film of the cell that allows to programme again do not cultivate the cell of programming again under the condition of sealing again.In also having another embodiment, before being inserted into ovocyte, the film of the cell that allows to programme again cultivates the cell of programming again under the condition of sealing again.

In also having another embodiment, by under the condition that allows chromatin to form, with the donor nuclei (for example in endogenous Protein virus nucleic acid have the nucleus that suddenly change and randomly have the nucleic acid of one or more coding heteroantibodies) of the substratum of programming again (for example cell extract) cultivation from cell of the present invention, and chromatin is inserted in the non-nucleus egg mother cell, thereby form first kind of embryo.From one or more cells of first kind of embryo and from one or more cells contacting in vitro fertilization, natural or second kind of embryo of parthenogenesis activated, generate the third embryo.Allowing the third fetal development to become under the condition of fetus the third embryo transfer in the uterus of host ungulate.In preferred embodiments, by allowing chromatin to form and do not causing under the condition of dna replication dna, will have the donor nuclei that is less than the quadruplet homologous chromosomes and contact with the substratum of programming again and form chromatin.Preferably, under the condition that allows chromatin to form, donor nuclei is contacted with following one or more: to resist-mitotic division extract, washing agent and/or salts solution during NuMA antibody existing or lack, perhaps protein kinase solution.

Use from the preferred embodiment aspect two kinds of embryos' the cells produce ungulate in any above-mentioned relating to, one of at least the first kind of embryo and second kind of embryo are fine and close embryo (compactionembryo).In another embodiment, first kind of embryo is in different cell stages with second kind of embryo.First kind of embryo and being used to produces second kind of embryo's donorcells can be from same species or from different genera or species.Preferably, at least 10,20,30,40,50,60,70,80,90,95 or 100% cell of trophectoderm of fetus or placenta tissue derives from second kind of embryo, and perhaps the cell of at least 30,40,50,60,70,80,90,95 or 100% in inner cell mass of fetus or the fetal tissue derives from first kind of embryo.In other preferred embodiments, first kind of embryo or the third embryo are with than having equal amts and from horizontal expression nuclear lamina protein A, lamin C or the NuMA albumen of at least 5 times of the expressed respective horizontal height of the contrast embryo of same species.

Use from the preferred embodiment aspect two kinds of embryos' the cells produce ungulate in any above-mentioned relating to, before the cells contacting that makes from various embryos, remove first kind of embryo or second kind of embryo zona pellucida partly or entirely.In one embodiment, will they be contacted from first kind of embryo and second kind of embryo's cell adjacently placed in solution or on the solid support.In another embodiment, with standard technique will injection cell to the second kind of embryo from first kind of embryo in.Can be Anywhere with this injection cell to the second kind of embryo, the embryo's periphery between zona pellucida and the embryo itself for example.Natural embryo comprise adopt standard method from conceived and ungulate (for example ox) to perform the operation or the vertical embryo who obtains of non-operation.Embryo in vitro fertilization comprises the interior sperm injection embryo of the kytoplasm that adopts standard method to produce.Can expect, can combined togetherly be formed for preparing the chimeric embryo of clone's ungulate from two or more embryos' cell (for example from three, four, five, six kind or more kinds of embryos' cell).

Produce the preferred embodiment of ungulate

In any preferred embodiment aspect above-mentioned, by enrichment or consume a kind of composition, for example dna methyltransferase, histone deacetylase enzyme, histone, protamine, lamin, transcription factor activator or repressor are modified the substratum of programming again (for example cell extract).In other preferred embodiments, in ovocyte or chimeric embryo, NuMA or the proteic expression level of AKAP95 are than the height at least 2 in the tenuigenin, 5,10 or 20 times in the nucleus.In also having other embodiments, the AKAP95 albumen of at least 30,40,50,60,70,80,90 or 100% in ovocyte or the chimeric embryo is with containing 0.1% Triton X-100,1mg/ml DnaseI, and the solution of the NaCl of 100mM or 300mM extracts.Preferably, before being inserted into non-nucleus egg mother cell, purifying chromatin from the substratum of programming again (for example extract).In another preferred embodiment, chromatin is inserted into non-nucleus egg mother cell comprises,, chromatin is contacted with the ovocyte with fusion gene compound allowing chromatin to enter under the condition of ovocyte.In also having another kind of preferred embodiment, the offspring that fetation becomes can survive.Preferably, at least 1,3,5,10,20,30,40,50,60,70,80 or 90% nuclear transplantation ovocyte or fetal development become the offspring that can survive.In this method, contain the chromatinic ovocyte or the cell of programming again allowing to cultivate under the condition of cytodifferentiation, and one of the cell that generates is cloned one or many again.Be used for donor nuclei, acceptor chromatin or the donorcells of present method and ovocyte and can be from same species or from different plant species or kind.Desirably, corresponding natural cell of the transgenic ungulates expression ratio of donorcells or generation or ungulate at least 10,20,40,60,80,90,95 or 100% functional protein or total protein less.Ovocyte is fertilization or unfertilized.Preferably, donor nuclei, chromatin or changed the cell of the cell of processing thoroughly from G phase or G0 phase.In addition, preferably the genomic dna with donorcells is identical basically for the genomic dna of clone's embryo, fetus or ungulate.It is also contemplated that, the cell of chromatin or programming again can be inserted into and produces chimeric embryo, fetus or ungulate among the embryo, they contain have with chromatin or the cell of the substantially the same DNA of cell that programmes again with have with the embryo in the mixture of cell of the substantially the same DNA of the cell of natural generation.It is also contemplated that tool nuclear ovocyte can be used in the method for the present invention.

The substratum of programming again that is used for any aspect of the present invention can contain or not contain heterologous nucleic acid.In other embodiment preferred, in nucleic acid in allowing carrier and the chromatinic genome under the condition of corresponding nucleic generation random integration or homologous recombination, chromatin in the substratum of programming again or that form in being changed the cell of processing is thoroughly contacted with the carrier of the nucleic acid with the interested gene of coding, chromatinic genome is changed.Owing to lack complete plasma membrane and lack nuclear membrane, in being changed the cell of processing thoroughly or solution in chromatin than the easier modification of the cell of natural generation.The example that can be used to produce the cell of the extract of programming again comprises embryonic stem cell and from the adult stem cell of brain, blood, marrow, pancreas, liver, skin or any other organ or tissue.Other exemplary cell extracts of programming again comprise ovocyte extract (for example ox or sea urchin egg parent cell extract) and male sex-cell extract (for example from vertebrates, invertebrates or Mammals for example spermatogonium, spermatocyte, spermatid or the sperm extract of ox).Donor or the cell of being changed processing thoroughly can be non-immortalization or natural, the spontaneous or hereditary infinite multiplications of going up.Donorcells, cell, recipient cell or the tenuigenin of being changed processing thoroughly can be from any stage sources, for example from embryo, fetus, childhood or Adult Mammals (for example ungulate).May accept less spontaneous mutation from the cell of originating, and after being inserted into ovocyte, may have the long life-span than stage morning.

Be used to breed the method for ungulate

In any above-mentioned preferred embodiment that is used for producing the method for ungulate or ungulate cell, the offspring that fetus with two or more genetic modifications maybe can survive is produced in ungulate of the present invention and another kind of ungulate (for example have the ungulate of sudden change or have the ungulate of nucleic acid of coding heteroantibody on heavy chain of antibody or light chain nucleic acid) mating.Preferably, from fetus or offspring, separate one or more cells, and in isolated cells, import one or more other genetic modifications.

Produce the method for antibody

The present invention also provides the method for producing antibody with the ungulate of the present invention of expressing heteroantibody (for example people's antibody).A kind of this method comprises the ungulate of the present invention that one or more interested antigen is administered to the nucleic acid with coding heteroantibody locus.Nucleic acid fragment in the locus is reset, and causes generating being specific to antigenic antibody.From ungulate, reclaim antibody.Antibody can be monoclonal or polyclonal, and preferably interested antigen is had reactive behavior.Preferably, from the serum of ungulate or milk, reclaim antibody.

In one aspect, the invention provides the method that another kind is used to prepare antibody, it comprises from the ungulate of the present invention of the nucleic acid with coding heteroantibody locus and reclaims heteroantibody.Nucleic acid fragment in the locus is reset, and causes generating being specific to antigenic antibody.Antibody can be monoclonal or polyclonal, and preferably interested antigen is had reactive behavior.Preferably, from the serum of ungulate or milk, reclaim antibody.

Nucleic acid

The present invention also provides the nucleic acid (for example carrier) of the prion gene in the ungulate (for example ox) that can be used for suddenling change.A kind of this nucleic acid has a kind of box, this box along 5 ' comprise having first homology zone, the positive selectable marker of the sequence substantially the same and have second homology zone with the substantially the same sequence in second zone of Protein virus nucleic acid with first zone of the endogenous Protein virus nucleic acid of ungulate to 3 ' direction.In one embodiment, first homology zone (for example less than 3,2,1.5 or the zone of 1kb) is shorter than second homology zone (for example at least 7,8,9 or the zone of 10kb), and this box can be integrated in the endogenous Protein virus nucleic acid of this cell.In another embodiment, first homology zone is longer than second homology zone.Preferably, the respective regions that will be suddenlyd change of the endogenous Protein virus nucleic acid in first and/or second homology zone and the cell is isogenic.

In a related aspect, the invention provides nucleic acid with box, this box comprises first homology zone, positive selectable marker with sequence substantially the same with first zone of the endogenous Protein virus nucleic acid of ungulate, have second homology zone with the substantially the same sequence in second zone of Protein virus nucleic acid, and the negative selection marker opposite (for example DT-A or Tk) (for example, promotor is connected to the short terminal negative selection marker in homology zone) with positive mark's direction.This box can be integrated in the endogenous Protein virus nucleic acid of cell.Desirably, the length in a homology zone is at least 7,8,9 or 10kb, and the length in another homology zone is less than 3,2,1.5 or 1kb.In some embodiments, partly or entirely negative selection marker is positioned at short homology zone.Preferably, the respective regions that will be suddenlyd change of the endogenous Protein virus nucleic acid in first and/or second homology zone and the cell is isogenic.

In yet another aspect, the invention provides the composition that comprises nucleic acid of the present invention and spermidine (for example 0.1-10mM spermidine).

The preferred embodiment of above-mentioned aspect

Preferably, antiserum(antisera) of ungulate or milk have polyclonal human normal immunoglobulin.Preferably, antiserum(antisera) or milk are from ox, sheep, pig or billy goat.In a further preferred embodiment, the antigen of the directed opposing expectation of Igs.In preferred embodiments, this antiserum(antisera) is used as the intravenously immunoglobulin (Ig) (IVIG) of treatment or prevention people's disease.In another preferred embodiment, interested antigen is applied to ungulate, and generates the antigenic Igs of directed opposing by ungulate.Preferably, in heteroimmunity globulin gene seat is reset the product nucleus acid fragment and with the heteroantibody that target antigen is had reactive behavior.Preferably, antiserum(antisera) and/or milk contain than

endogenous antibody height

2,5,10,20 or 50 times heteroantibody at least, perhaps do not contain endogenous antibody.If desired, can produce hybridoma and monoclonal antibody with the xenogenesis B cell that derives from above-mentioned transgenic ungulates (for example transgenic cattle).It is also contemplated that, can then carry out chemically modified, make it covalently to be connected on a kind of toxin, therapeutical active compound, enzyme, cytokine, radio-labeling, fluorescent mark or the affinity labeling isolating heteroantibody from ungulate (for example people's antibody).If desired, this fluorescent mark or radio-labeled can be used to external or intravital antibody imaging.

Preferably, the Protein virus nucleic acid mutation has reduced the generation of the prion protein of infectious form.In preferred embodiments, the content of the prion protein of the infectious form that is generated by ungulate lacks 80,70,60,50,40,30,20,10 or 5% than the content of the prion protein of the contrast ungulate generation of not having sudden change.Preferably, do not produce infectious prion protein.

In preferred embodiments, sudden change be insert from the Protein virus nucleic acid (for example coding region) of other animals (for example different ungulate kind or kinds) partly or entirely.Preferably, this xenogenesis Protein virus nucleic acid substitutes the part or all of of endogenous Protein virus nucleic acid.In preferred embodiments, xenogenesis Protein virus nucleic acid is from kind or kind that prion-infected resistivity is improved, for example sheep of antipruritic disease.Preferably, the prion-infected ratio of ungulate with sudden change does not have the prion-infected ratio low 80,70,60,50,40,30,20,10 or 5% of ungulate of sudden change.

Preferred ungulate and donorcells

Ungulate comprises the member of Perissodactyla (Perissodactyla) and Artiodactyla (Artiodactyla), for example any member of Bos (Bos).Other preferred ungulates comprise sheep, big angle sheep, goat, bison, antelope, ox, horse, donkey, mule, deer, elk, caribou, buffalo, camel, yamma, alpaca, pig and resemble.

The cell that preferred donorcells comprises differentiation is epithelial cell, neurocyte, epidermic cell, keratinocyte, hematopoietic cell, melanoma cells, chondrocyte, B-lymphocyte, T-lymphocyte, red corpuscle, scavenger cell, monocyte, inoblast and muscle cell for example; Undifferentiated cell is embryonic cell (for example stem cell and embryonic genital cell) for example.In another preferred embodiment, this cell is from female repro ductive system, for example mammary gland, ovary, granulosa cell or oviduct cell.Other preferred cells comprise fetal cell and placenta cells.Preferred cell also comprises those from any organ, for example the cell in bladder, brain, esophagus, uterine tube, heart, enteron aisle, gall-bladder, kidney, liver, lung, ovary, pancreas, prostate gland, spinal cord, spleen, stomach, testis, thymus gland, Tiroidina, tracheae, urinary catheter, urethra and uterus.Preferably, the ovocyte of donorcells, donor nuclei, donor chromatin or reconstruct is not a tetraploid.

In also having another embodiment preferred, nucleus, change the cell of processing or karyomit(e) from transgenic cell or ungulate or contain and be not present in donorcells or be not present in sudden change in the n cell thoroughly.Preferably, transgenosis donor nuclei and donorcells coding in the clone ungulate makes it the protein to disease or the raising of parasitic resistance.Selectively, donor nuclei or donorcells are made the ungulate of DCRP generate recombinant products by through engineering approaches, for example produce people's albumen in urine, blood or the milk of ox.For example, by being inserted in hematuria platelet lysin (uroplakin) the promotor control proteic polynucleotide sequence of coding people down, marking protein in the urine of ox.The therapeutic protein that can generate in the milk of clened cows comprises human blood coagulation for example any (Voet and Voet, Biochemistry, the John Wiley ﹠amp of factor I-X III; Sons, New York, 1990).These heterologous proteins can be in the prolactin promotors or are adapted at expressing under the control of any other promotor of expressing in the cow's milk juice.Can come purifying from the recombinant protein of these and other tissues or liquid (referring to, Ausubel etc. for example, with above) with the purification process of standard.

Definition

As used in this, " artificial chromosome " is meant Mammals karyomit(e) or its fragment, and it has manually modified, but for example increases a selective marker, increase cloning site, a kind of or a plurality of Nucleotide of deletion, the one or more Nucleotide of replacement etc." human artificial chromosome " or " HAC " is meant the artificial chromosome that derives from one or more human chromosomes.Artificial chromosome can be independent of host cell the intrinsic staining body be maintained in the host cell.In this case, HAC duplicates with being stabilized and is separated with the intrinsic staining body.Alternately, HAC is transferred to or is inserted in the intrinsic staining body of host cell.Two or more artificial chromosomes can simultaneously or in a sequence be inserted in the host cell.For example, can import the artificial chromosome that derives from human chromosome #14 (comprising the Ig heavy chain gene), human chromosome #2 (comprising Ig κ chain gene) and human chromosome #22 (comprising Ig λ chain gene).Selectively, can import the artificial chromosome that comprises xenogenesis Ig heavy chain and Ig light chain gene, for example Δ HAC or Δ Δ HAC.Preferably, the heavy chain gene seat is positioned on the different chromosome arms with the light chain gene seat and (promptly is positioned on the centric not ipsilateral).In also having other embodiment preferred, the size of whole HAC less than or approximate 10,9,8 or 7Mb.

" arrange or do not reset the nucleic acid of form in advance " and be meant the nucleic acid that V (D) J reorganization does not take place.In preferred embodiments, by one or more nucleic acid, whole nucleic acid fragments of the V gene fragment of encoding antibody light chain all nucleic acid fragments with coding J gene fragment are separated.Preferably, by one or more nucleic acid, whole nucleic acid fragments of the V gene fragment of encoding antibody heavy chain all nucleic acid fragments with the encoding D gene fragment are separated, and/or by one or more nucleic acid, whole nucleic acid fragments of encoding D gene fragment all nucleic acid fragments with the J gene fragment of encoding are separated.Preferably, the nucleic acid that is not rearranged form is the people basically.In other preferred embodiments, it is 70,80,90,95 or 100% identical that this nucleic acid and respective regions from people's natural acid have at least.

The content and/or the activity of endogenous antibody " reduce " is meant the endogenous that reduction generates by B cell or B cell colony, the content of functional antibodies, as the U.S.S.N.10/441 of application on May 19th, 2003, described in 503.This reduction of endogenous antibody may be because the endogenous antibody content that each B cell produces descends, has the endogenous B cell quantity of function to descend or their combinations.Preferably, by B emiocytosis or expressing or the content of the endogenous antibody of the bone-marrow-derived lymphocyte surface expression of secretion endogenous antibody reduces at least 25,50,75,90 or 95%.In another preferred embodiment, come the mammiferous sample of autoreceptor for example the quantity of the endogenous B cell in the blood sample reduced at least 25,50,75,90 or 95%.

" bifunctional antibody " is meant the antibody that comprises on the different fragments that covalently is attached to different antibodies or antibody or the antibody of antibody fragment.In a preferred embodiment, antibody or fragment are attached on the different epi-positions that manifest on the same antigen.Other preferred bifunctional antibodies are attached on two kinds of different antigens, for example are attached on the light chain and heavy chain of antibody of antibody.The Protocols in Molecular Biology of standard is those technology described here two nucleic acid that are used to be operably connected for example, make integrative nucleic acid a kind of bifunctional antibody of encoding.

" fragment " is meant the polypeptide with successive amino acid region, but this successive amino acid region is identical shorter than full length sequence with the respective regions of antibody of the present invention.Based on standard analysis, for example described here those, this fragment can be as corresponding antibodies in conjunction with identical antigen.Preferably, this fragment is at least corresponding antibodies and antigenic bonded 20,40,60,80 or 90% with antigenic the combination.

" purifying " is meant from separating natural other components that accompany with it.Typically, when not containing the natural with it protein that accompanies, antibody and naturally occurring organic molecule, when a kind of composition accounted at least 50% on weight, this composition was pure basically.Preferably, this composition is at least 75% on weight, more preferably is at least 90% and most preferably be at least 99% pure.Can be by chemosynthesis, separate this composition from natural origin, perhaps in the host cell that can not generate this composition naturally of reorganization, generate this one-tenth and assign to obtain pure basically composition.Those methods that those skilled in the art can adopt standard method for example to be described by (with above) such as Ausubel are come protein purification, carrier and organoid.Preferably than starting materials pure at least 2,5 or 10 times of this compositions adopt polyacrylamide gel electrophoresis, column chromatography, optical density(OD), HPLC analyzes or western blot analysis (Ausubel etc., with above) is measured.Preferred purification process comprises immunoprecipitation, column chromatography such as immunoaffinity chromatography, magnetic bead immune affinity purifying and uses and the elutriation of dull and stereotyped bonded antibody.

" chromatin " is meant the karyomit(e) of more than one not tunicle bag quilt.Preferably, chromatin contains all karyomit(e)s of cell.Can programme again in the substratum (for example mitotic division extract) by nucleus being exposed to mitotic division described herein, form the chromatin that contains spissated chromosomal artificial induction.Alternatively, nucleus can be exposed to one of following generate to contain concentrate or the chromosomal artificial induction's of partial concentration chromatin, as described herein: contain mitotic division extract, washing agent and/or the salts solution of anti--NuMA antibody, perhaps protein kinase solution.It is not the discrete karyomit(e) of body contact each other that chromatin may contain, and perhaps may contain the karyomit(e) of two or many body contacts.

If desired, can adopt standard method, detect chromosomal concentrating degree by the coloring degree of measuring DNA dyestuff DAPI.When karyomit(e) concentrated, this dye levels increased.Therefore, the coloring degree of the most spissated karyomit(e) (being appointed as 100% concentrates) of the condensation of chromosomal coloring degree and interval (being appointed as 0% concentrates) and m period relatively.Based on this comparison, can determine maximum spissated ratio.Preferred spissated chromatin concentrates at least 50,60,70,80,90 or 100%.Preferably go the chromatin of concentrated or partial concentration to concentrate for being less than 50,40,30,20 or 10%.

" nucleus " is meant the major part that contains cell or the film restrictive cell device of all DNA.DNA is wrapped in the karyomit(e) to go spissated form.Preferably, bag is comprised one deck or two-layer lipid or has nucleoprotein by the film of DNA.

" have the nucleus of quadruplet homologous chromosomes at least " and be meant to have the nucleus of the DNA concentration of 4n at least, wherein " n " is the normal haploid chromosomes group that is present in the Mammals of specific kind or kind.This nucleus does not have four parts of range genes or locus.Preferably, nucleus is double, therefore has two cover homologous chromosomess, but is less than two pairs of complete chromatids.

" protokaryon " is meant the haploid cell nuclear that is produced by mitotic division or nuclear transplantation protokaryon.Female pronucleus is the nucleus before ovocyte or ovum and the male pronucleus fusion.Male pronucleus is still to examine with female pronucleus fusion spermatid before after the time of fertilization enters ovocyte or ovum.The nuclear transplantation protokaryon is donorcells, nucleus or chromatin to be imported the protokaryon (for example amphiploid protokaryon) that forms behind the ovocyte.The nuclear transplantation protokaryon has the homologous chromosomes that is less than quadruplet.

" donorcells " is meant the cell that nucleus or chromatin come from or changed the cell of processing thoroughly.

" change processing thoroughly " and be meant and on plasma membrane, form the hole or partially or completely remove plasma membrane.

" substratum of programming again " is meant that permission removes a kind of composition or will add cell, nucleus, chromatin or chromosomal solution from the composition of solution from cell, nucleus, chromatin or karyomit(e).Preferably, the interpolation of composition or remove to improve or reduced donorcells, chromatin or nucleus or contained again chromatin or the mRNA in the nuclear cell or the protein expression level of programmingization.In another embodiment, in the substratum of programming again, cultivate cell, chromatin or the nucleus changed processing thoroughly changed the cell of being changed processing thoroughly or contain again programmingization chromatin or with the phenotype of the nuclear cell of donorcells phenotypic correlation.In also having another embodiment, in the substratum of programming again, cultivate cell, chromatin or the nucleus of being changed processing thoroughly, make and changed the cell of processing thoroughly or contain programme again chromatin or nuclear cell acquisition or the forfeiture activity relevant with donorcells.

The exemplary substratum of programming again comprises and does not contain for example solution of protein or nucleic acid of biomolecules, as damping fluid.This solution can be used for removing one or more compositions from nucleus, chromatin or karyomit(e).Other substratum of preferably programming again are extract, for example from nucleus, tenuigenin or their bonded cell extract.Exemplary cell extract comprises from ovocyte (for example ovocyte of Mammals, vertebrates or invertebrates), male sex-cell (for example the sexual cell of Mammals, vertebrates or invertebrates for example spermatogonium, spermatocyte, spermatid or sperm), and the extract of stem cell (for example growing up or embryonic stem cell).Also having other substratum of programming again is the solution or the extract of the composition that adds one or more natural or reorganization (for example nucleic acid or protein for example dna methyltransferase, histone deacetylase enzyme, histone, protamine, lamin, transcription factor, activator, repressor, chromatin reconstitution albumen, somatomedin, interleukin-, cytokine or other hormones), perhaps removes the extract of one or more compositions.Also have other the substratum of programming again to comprise washing agent (0.01%-0.1% for example, 0.1%-0.5% or 0.5%-2% ionic or non-ionic type washing agent, one or more following washing agent: SDS for example, TritonX-100, TritonX-114, CHAPS, Sodium desoxycholate, the n-octyl glucoside, NonidetP40, IGEPAL, Tween 20, Tween 40 or Tween 80), salt (for example, about 0.1,0.15,0.25,0.5,0.75,1,1.5 or 2M NaCl or KCI), polyamines (for example, about 1 μ M, 10 μ M, 100 μ M, 1mM or 10mM spermine, spermidine, protamine or poly-lysine), protein kinase (for example, the kinases 1 that cyclin relies on, protein kinase C, protein kinase A, map kinase, the kinases that calcium/calmodulin relies on, CK1 casein kinase or CK2 casein kinase), and/or inhibitors of phosphatases (for example, about 10 μ M, 100 μ M, 1mM, 10mM, 50mM, in the following inhibitor of 100mM one or more: sodium orthovanadate, trisodium phosphate, Sodium Fluoride, NIPP1, inhibitor 2, PNUTS, SDS22, AKAP149 or ocadaic acid).In some embodiments, the substratum of programming again contains anti--NuMA antibody.If desired, can simultaneously or use the multiple substratum of programming again programme again donorcells, nucleus or chromatin continuously.

" interval programme substratum " is meant and induces chromatin to go to concentrate and solution that nuclear envelope forms (for example interval cell extract) again.

" mitotic division programme substratum " is meant and induces chromatin to concentrate and nuclear envelope disruptive solution (for example mitotic cell extract) again.

" cell of programming again " is meant the cell that is exposed in the substratum of programming again.Preferably, have at least 1,5,10,20,25,50,75,100,150,200,300 or more kinds of mRNA or protein molecule in the cell of programming again, expressed, these mRNA or protein are at donorcells or changed thoroughly in the cell of processing and do not expressed.In another preferred embodiment, in the cell of programming again, express and not at donorcells or the quantity of being changed the mRNA that expresses in the cell of processing or protein molecule thoroughly between 1 to 5, between 5 to 10, between 10 to 25, between 25 to 50, between 50 to 75, between 75 to 100, between 100 to 150, between 150 to 200 or between 200 to 300, comprise endpoint value.Preferably, at least 1,5,10,20,25,50,75,100,150,200,300 or more mRNA or protein molecule at donorcells or changed thoroughly in the cell of processing and expressed, and in the cell of programming again, do not expressed.In a further preferred embodiment, at donorcells or the quantity of being changed the mRNA that expresses in the cell of processing and in the cell of programming again, do not express or protein molecule thoroughly between 1 to 5, between 5 to 10, between 10 to 25, between 25 to 50, between 50 to 75, between 75 to 100, between 100 to 150, between 150 to 200 or between 200 to 300, comprise endpoint value.In also having another embodiment preferred, these mRNA or protein molecule are expressed in the donorcells (for example donor or the initiator cell changed thoroughly) and the cell of programming again, but by standard method (referring to, Ausubel etc. for example, together above) measure, the expression level in these cells differs at least 2,5,10 or 20 times.

" add a kind of factor " and be meant composition that a kind of factor is attached to chromatin, karyomit(e) or nuclear envelope for example on nuclear membrane or the nuclear matrix.Selectively, this composition is transported in the nucleus, makes it by nuclear envelope combination or bag quilt.Preferably, the content that is incorporated on the karyomit(e) or is positioned the composition on the nucleus has increased at least 25,50,75,100,200 or 500%.

" remove a kind of factor " and be meant from the composition of chromatin, karyomit(e) or nuclear envelope nuclear membrane or nuclear matrix a kind of factor of dissociating for example.Selectively, this factor is transported to outside the nucleus, makes it no longer by nuclear envelope combination or bag quilt.Preferably, the content that is incorporated on the karyomit(e) or is positioned the factor on the nucleus has descended at least 25,50,75,100,200 or 500%.

" a kind of enrichment of the factor or consumption " be meant add or remove the original factor that is present in natural existence in the substratum of programming again (for example cell extract) or reorganization content at least 20,40,60,80 or 100%.Selectively, can add the factor that nature is not present in the natural or reorganization in the substratum of programming again.The preferred factor comprises protein for example dna methyltransferase, histone deacetylase enzyme, histone, protamine, lamin, transcription factor, activator and repressor; Membrane vesicle and organoid.In a preferred embodiment, as described below, before being added to the substratum of programming again, this composition is purified.Selectively, one of purification process as described below is used to remove a kind of factor of not expecting from the substratum of programming again.

" clone again " and be meant that being used to second takes turns the clone.Especially, come the cell of embryo, fetus or the adult of free method production of the present invention in substratum (for example mitotic cell extract) is programmed in mitotic division again, to cultivate, generate the chromatin that is used for being inserted into non-nucleus egg mother cell, as mentioned above.Selectively, as mentioned above, this cell is changed thoroughly, cultivates in the substratum of programming again, and is inserted in the non-nucleus egg mother cell.Carry out two-wheeled or many wheel clones, improve in the end a probability of taking turns the offspring that clone's back generation can survive thereby cause that donor chromatin or donorcells are carried out extra programming again.

" offspring that can survive " is meant the Mammals of survival outside the uterus.Preferably, after leaving the parent host, this Mammals has survived 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 6 months, 1 year at least.This Mammals does not need the recycle system of uterus environment to survive.

" nuclear transplantation ovocyte " or " consideration convey moves ovocyte " are meant insertion or have merged donorcells, nucleus or chromatinic ovocyte.The embryo who is formed by this ovocyte is known as " nuclear transplantation " or " consideration convey moves " embryo.

" embryo " or " embryo's " is meant the tenuigenin of growing in not implanted parent host's also the uterine endometrium.Therefore, " embryo " is meant the ovocyte of fertilization; The ovocyte that contains donor chromatin, nucleus or the cell of programming again; The tenuigenin of growing before blastula stage; Or it is any before the uterine endometrium of implant carrier receptor body and other tenuigenin of growing of the etap before forming gonocrista.The embryo may represent a plurality of cell development stages.For example, a cell stage can be known as zygote; The solid spherical tenuigenin that is produced by the embryo of the spilting of an egg can be known as morula, can be known as blastaea and have blastocelic embryo." embryonic cell " be separate from or be included in cell among the embryo.

" derive from embryo's cell " and be meant the cell that produces by intraembryonic cytodifferentiation.

" chimeric embryo " is meant by the plastidogenetic embryo from two or more embryos.The fetus or the offspring that generate have the cell that only derives from one of initial embryo or derive from a not only initial embryo's cell.If desired, can adopt the fish analysis of standard or, determine to be attached to the ratio in placenta tissue and the fetal tissue from each embryo's cell to adding a painted analysis of the film on the embryo to.

" chimeric ungulate " is meant by the plastidogenetic ungulate from two or more embryos.This ungulate can have the cell that only derives from one of initial embryo or derive from a not only initial embryo's cell.If desired, can adopt the fish analysis of standard or, determine to be attached to the ratio in placenta tissue and the fetal tissue from each embryo's cell to adding a painted analysis of the film on the embryo to.

" fine and close preceding embryo " is meant fine and close embryo before.In fact, the preceding embryo of densification is not at the surface expression E-of its blastomere cadherin.Preferred fine and close preceding embryo expresses and lacks 3,5,10,20,30 or 40 times E-cadherin at least than the complete densification embryo of same species.

" fine and close embryo " is meant the embryo of taking place after densification or the densification.Fine and close embryo's blastomere is expressed the E-cadherin in its surface.Can adopt anti-E-cadherin antibody to measure the expression of this E-cadherin with standard method.The E-cadherin increases the adhesion between the blastomere.Preferred fine and close embryo comprises the embryo that compaction process is finished.The E-cadherin that fine and close preceding embryo's height of other preferred fine and close embryo's expression ratio identical type is at least 3,5,10,20,30 or 40 times.

" fetus " is meant the tenuigenin of growing in the uterine endometrium of implanting the parent host.Fetus has for example gonocrista of the easy special characteristic of being determined by those skilled in the art." fetal cell " be from or be included in any cell in the fetus.

" parthenogenesis " or " parthenogenesis activation " is meant that its nucleus does not merge the ovocyte that forms zygote or the growth of ovum with male pronucleus.For example, unfertilized ovocyte can be induced differentiation.

" zona pellucida " is meant transparent, the resilient acellular layer that is looped around outside many mammiferous ovocytes or the ovum.

" trophectoderm " is meant in the blastula stage that mammal embryo is grown around blastocelic outermost cellular layer.In the growth afterwards, trophectoderm forms the most of or whole of placenta tissue.

" inner cell mass " is meant the cell that is surrounded by trophectoderm.In the growth afterwards, the inner cell mass cell forms the major part of fetal tissue.

" be specific to a kind of mRNA or protein of cell type " and be meant the mRNA or the protein of expressing with certain level in a kind of cell type, this level is than at least 10,20,50,75 or 100 times of the expression level height in the every other cell type.Preferably, mRNA or protein are only expressed in a kind of cell type.

" sudden change " is meant the change in natural or reference nucleotide sequence, for example inserts, deletion, phase shift mutation, silent mutation, nonsense mutation or missense mutation.Preferably, the aminoacid sequence by nucleic acid sequence encoding has the amino acid change that at least one is different from native sequences.The example that is used for changing the DNA recombinant technology of cell, embryo, fetus or mammiferous genome sequence comprises the dna sequence dna from another kind of organism (for example people) is inserted into genome, disappearance one or more dna sequence dna and imports one or more base mutations (for example fixed point or random mutation) in the target dna sequence dna.The example that produces the method for these modifications comprises that retrovirus insertion, artificially colored body technique, gene insert, insert at random tissue-specific promoter, homologous recombination, gene targeting, Transshipment Permitted element and any other is used to import the additive method of foreign DNA.All these methods are (referring to, the Ausubel etc. for example, with above) that knows for the technician of biology field.Chromatin, karyomit(e) and can be used to method of the present invention from the nucleus that contains by the transgenic cell of modifying DNA or donor transgenic cell.

" infinite multiplication " is meant and can takes place than with the natural control cells of infinite multiplication cell same cell type, kind and species or than the donorcells height at least 25,50,75,90 of this infinite multiplication cell of deriving or 95% cytodifferentiation.Preferably, the infinite multiplication cell takes place than control cells height 2,5,10 or 20 times differentiation at least.More preferably, the infinite multiplication cell can carry out unlimited cytodifferentiation.The infinite multiplication cell comprises naturally to be needed in the body or the cell of external sudden change, and this sudden change changes its normal growth regulating process.Also have other preferred infinite multiplication cells to comprise and express for example cell of ras, myc, abl, bcl2 or neu of proto-oncogene by genetic modification, perhaps with transfering DNA or the RNA viruses cell (Kumar etc. of Epstein-Barr virus or SV40 virus infection for example, Immunol.Lett.65:153-159,1999; Knight etc., Proc.Nat.Acad.Sci.USA 85:3130-3134,1988; Shammah etc., J.Immunol.Methods 160-19-25,1993; Gustafsson and Hinkula, Hum.Antibodies Hybridomas 5:98-104,1994; Kataoka etc., Differentiation 62:201-211,1997; Chatelut etc., Scand.J.Immunol.48:659-666,1998).Can also express telomerase gene (Roques etc., Cancer Res.61:8405-8507,2001) by genetically modified cell.

" non-infinite multiplication " is meant the infinite multiplication that is not above-mentioned.

" gene fusion compound " is meant when being positioned at adjacent cells, improves the compound that chromatin or nucleus are inserted into the probability in the recipient cell that for example the gene fusion compound can improve the affinity of chromatin or nucleus pair cell plasma membrane.The gene fusion compound also may promote nuclear nuclear membrane to be connected with the plasma membrane of cell.

" substantially the same " is meant to have and another sequence at least 60,70,80,90 or 100% identical sequence.Usually adopt sequence analysis software, measure sequence identity (the sequence analysis software bag of Genetics Computer Group for example with its specific default parameters, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, WI 53705).This software mates similar sequence by giving the homology degree to various replacements, deletion and other modifications.

Advantage

The invention provides and a large amount of on one or two allelotrope of prion gene, have the relevant advantage of transgenic ungulates (for example ox) of sudden change with producing.For example, Protein virus knockout carrier described here, cell culture processes and transfection method are with the prion gene in the wonderful high frequency deactivation ox cell.A large amount of correct donorcellses of practicing shooting and improved nuclear transplantation method of the present invention (for example, chromatin shifts and SLOT) are very easy to low statement or do not express the production of the cloned, transgenic ungulate (for example ox) of prion protein.The transgenic ungulates of these generations is expectation sources of agricultural-food (for example meat) and the medicine therapeutic antibodies of one or more heterologous nucleic acid expressing human in the transgenic ungulates (for example by).

According to following detailed description and claims, other features and advantages of the present invention are obvious.

Brief description

Present specification contains color drawings (accompanying drawing 31,34A-34C, 36A, 36B, 42A-42D, 45A, 46D, 47A, 47C, 48,49B, 50A-50D and 51C-51E).Will the providing as requested and after paying suitable expense of copy with this patent of color drawings or patent disclosure by Patent Office.

Accompanying drawing 1A has described to be used to produce and has contained Ig and knock out general introduction with the method for human artificial chromosome's cow.Time line among the accompanying drawing 1A is based on following estimation: be used to prepare the Ig knockout carrier in 18 months and production knocks out cell, be used in 2 months generating fetus by the described cell that knocks out, be used to carry out follow-up knocking out in 9 months, the gestation farrowing needs 9 months, before producing the embryo, there are 12 months, were used to carry out HAC and shift in 6 months by calf.

Accompanying drawing 1B has described and has been used for producing the general introduction of method that contains sudden change and contain the cow of Δ HAC or Δ Δ HAC at endogenous Ig gene.For the time line among Figure 1B, estimate to screen 250 colonies weekly, in 3 months, screen 3,000 colonies altogether, so that separate the male and female cell that knocks out.Suppose that per 1,500 bacterium colony produces one or more colonies that knock out.The ungulate that knocks out of isozygotying can produce by following method: (1) is before nuclear transplantation, isolating knocking out in the cell introduced in second Ig sudden change, (2) in the offspring's who derives from embryo, fetus (for example pregnant about 60 days fetus) or produce cell, introduce second Ig sudden change by first round nuclear transplantation, and as second donorcells of taking turns nuclear transplantation, perhaps (3) allow the ungulate mating with the resulting cell that isozygotys.In accompanying drawing 1A and 1B, " together " expression is isozygotied; " partly " expression is hemizygous; " H " represents heavy chain; " L " represents light chain; " HAC " represents the human artificial chromosome; " HAC1 " represents any HAC; And second kind of HAC of " HAC2 " expression.

Accompanying drawing 2A comprises according to μ of the present invention (IgM heavy chain) and knocks out construct.Accompanying drawing 2B is the restriction map from the immunoglobulin loci of Holstein ox (Holstein).

Accompanying drawing 3A-3B comprises the synoptic diagram of construct pSTneoB and pLoxP-StneoB.Be respectively applied for preparation μ knockout dna construct.

Accompanying drawing 3C describes μ knockout dna construct.

Accompanying drawing 3D is described in the polynucleotide sequence (SEQ ID NO:47) that μ knocks out the 1.5kb district of the genome ox μ heavy chain gene seat that is used as first homologous region in the construct.

Accompanying drawing 3E is described in the polynucleotide sequence (SEQ ID NO:48) that μ knocks out the 3.1kb district of the genome ox μ heavy chain gene group that is used as second homologous region in the construct.In this sequence, each " n " represents any Nucleotide or do not have Nucleotide.Successive " n " Nucleotide district represents its polynucleotide sequence district of undeterminate about 0.9-1.0kb still.

The ox μ heavy chain that accompanying drawing 3F describes the tetracycline resistance knocks out construct.

Accompanying drawing 3G has described the polynucleotide sequence (SEQ ID NO:60) of ox K light chain DNA.The all or part of κ of the being used to light chain of this sequence knocks out in the construct.In addition, this K light chain can be used for separating and be used for the genome κ sequence of light chain that the K light chain knocks out construct.

Accompanying drawing 4 is the synoptic diagram that make up Δ HAC and Δ Δ HAC.

Accompanying drawing 5 is photos of sepharose, shows the genomic dna that has coding people's heavy chain and light chain in Δ HAC fetus.

Accompanying drawing 6 is photos of sepharose, shows C μ exon 3 in 77 days Δ HAC fetus (fetus #5996) of gestation and 4 expression.

Accompanying drawing 7 is sepharose photos, shows the rearrangement of endogenous Niu Chonglian in Δ HAC fetus #5996.

Accompanying drawing 8 is sepharose photos, shows the expression of people's heavy chain in Δ HAC fetus #5996 of rearrangement.

Accompanying drawing 9 is sepharose photos, shows the expression of montage constant region in Δ HAC fetus #5996 from people's heavy chain gene seat.

Accompanying drawing 10 is sepharose photos, shows the expression of people's heavy chain in Δ HAC fetus #5996 of rearrangement.

Accompanying drawing 11A is the polynucleotide sequence (SEQ ID NO:49) from the rearrangement people heavy chain transcript of Δ HAC fetus #5996.

Accompanying drawing 11B is a district (" search sequence ") of this sequence and the comparison (being respectively SEQID NO:50 and 51) of the anti-streptococcus pneumoniae antibody of people (" sequence to be measured ").For for the search sequence of Δ HAC fetus #5996, only show those Nucleotide different with the corresponding nucleotide of the anti-streptococcus pneumoniae antibody sequence of people.

Accompanying drawing 12A-12B describes from the two other polynucleotide sequence (SEQ ID NO:52 and 54) of the rearrangement people heavy chain transcript of Δ HAC fetus #5996 and the aminoacid sequence of deriving (being respectively SEQ ID NO:53 and 55) thereof.

Accompanying drawing 13 is sepharose photos, proves that Δ Δ HAC fetus #5580 contains people's heavy chain and light chain immunoglobulin loci.

Accompanying drawing 14 is sepharose photos, proves to contain people's heavy chain and light chain immunoglobulin loci among Δ Δ HAC fetus #5442A and the 5442B.

Accompanying drawing 15 is sepharose photos, shows the expression of montage μ constant region in Δ Δ HAC fetus #5442A from people's heavy chain gene seat.

Accompanying drawing 16 is sepharose photos, shows rearrangement and the expression of people's heavy chain gene seat in Δ Δ HAC fetus #5868A.

Accompanying drawing 17 is sepharose photos, shows rearrangement and the expression of people Ig λ locus in Δ Δ HAC fetus #5442A and 5442B.

Accompanying drawing 18 is sepharose photos, shows rearrangement and the expression of people Ig λ locus in Δ Δ HAC fetus #5442A.

Accompanying drawing 19 is sepharose photos, shows rearrangement and the expression of people Ig λ locus in Δ Δ HAC fetus #5868A.

Accompanying drawing 20 is described polynucleotide sequence and the aminoacid sequence of deriving accordingly (being respectively SEQ ID NO:56 and 57) from people's light chain transcript of the rearrangement of Δ Δ HAC fetus #5442A.

Accompanying drawing 21 is described from the polynucleotide sequence of people's light chain transcript of the other rearrangement of Δ Δ HAC fetus #5442A and corresponding deduced amino acid (being respectively SEQ IDNO:58 and 59).

Accompanying drawing 22A-22H is the facs analysis figure of Δ Δ HAC fetus #5442A (accompanying drawing 22A-22D) and 5442B (accompanying drawing 22E-22H) expressing human lambda light chain and ox heavy chain protein.Make from the anti-ox IgM antibody (accompanying drawing 22D and 22H) of the anti-people λ antibody (accompanying drawing 22C and 22D) of the lymphocyte of the spleens of these fetuses and phycoerythrin mark, FITC mark or do not have antibody (accompanying drawing 22A, 22B (22E and 22F)) and react, on the FASCalibur cell sorter, analyze then.A kind of percentage of cell of described antibody labeling wherein is presented at the below of each histogram.

Accompanying drawing 23 is the synoptic diagram of α-(1,3)-galactosyltransferase knockout carrier, and described carrier is used for puromycin resistance gene and transcription termination sequence are inserted in endogenous α-(1,3)-galactosyltransferase gene of ox cell.

Accompanying drawing 24 is for containing

exon

2,3 and 4, as the segmental synoptic diagram of BamHI-XhoI of described AAV targeting vector skeleton.The neomycin resistance mark is used for by being inserted into exon 4 described locus being inserted mutagenesis.Indicated the position in the annealing site of the PCR primer that is used for verifying subsequently correct target practice.

Accompanying drawing 25 is the synoptic diagram that make up the adeno associated virus construct that is designed for the endogenous IgH sequence of removing ox.

Accompanying drawing 26 is photos of sepharose, shows the pcr analysis of each transduction clone of correct target practice incident.The carrier that uses in this test is shown in the accompanying drawing 24.The PCR that indicating correct is practiced shooting inserts with the asterisk mark.

Accompanying drawing 27 is forms of listing the embryo's who carries HAC pregnancy rate.

Accompanying drawing 28A-28J is from having injected the facs analysis photo that anti-ox IgM antibody (das6) suppresses the peripheral blood lymphocyte of B cytocerastic test fetus or contrast fetus.Accompanying drawing 28A-28E is the facs analysis photo that comes directed opposing to carry out at the IgM of B cell surface expression molecule with anti-ox IgM antibody.Shown in accompanying drawing 28A and accompanying drawing 28B, about 19.82-26.61% expresses IgM from the peripheral blood lymphocyte of contrast fetus.On the contrary, 7.78, %, 11.80% or 3.95% expresses IgM (being respectively accompanying drawing 28C-28E) from three peripheral blood lymphocytes of having injected the fetus of anti-ox IgM antibody.Accompanying drawing 28F-28J is the facs analysis photo that comes directed opposing to carry out at the light chain of antibody molecule of B cell surface expression with anti-ox light chain antibody (das10).Shown in accompanying drawing 28F and accompanying drawing 28G, about 12.43-29.47% is from the peripheral blood lymphocyte expressing antibodies light chain molecule of contrast fetus.On the contrary, 2.54%, 13.77% or 3.99% from three peripheral blood lymphocyte expressing antibodies light chain molecules (being respectively accompanying drawing 28H28J) of having injected the fetus of anti-ox IgM antibody.

Nuclear envelope and and stromatin among the embryo of accompanying drawing 29A-29B diagram immunodetection before the implantation uterus of ox.Accompanying drawing 29A is the picture with the ox embryo in vitro fertilization of the protokaryon phase of same antibody inspection and 8-cell stage.Anti--the NuMA and the anti--AKAP95 of the mark in the female pronucleus of pronuclear-stage embryos of the arrow points among the accompanying drawing 29A.Illustration among the accompanying drawing 29A is with the DNA picture of 0.1 μ g/mlHoechst33342 mark (grating (bars), 20 μ m).Accompanying drawing 29B is the immunoblotting assay to ox inoblast (row of going up) and protokaryon embryo (row down).The molecular weight marker thing is shown in the right of accompanying drawing 29B with kDa.

Accompanying drawing 30 is ox donor inoblasts (donorcells), be in nuclear transfer embryo, the nuclear transfer embryo of protokaryon phase (merging back 19 hours) and as the photo of activated parthenogenesis protokaryon embryo described here of the chromatin enriching stage (merging back 3 hours) before ripe.Use anti-lamin B, Lamin A/C, NuMA and AKAP95 antibody in injection " hpi " back 3 hours ripe prochromatin enriching stage (" PCC ") and the decomposition of injection (" NTPN ") monitoring in back 7 hours donorcells nuclear and the assembling of new protokaryon.Use 10mM SrCl ₂The ovocyte of parthenogenesis activated M II phase is activating the female pronucleus (" Parth.PN ") of handling analysis formation in back 5 hours.In the nuclear transplantation in cattle embryo's of protokaryon phase protokaryon, assemble Lamin A/C.With 0.1 μ g/mlHoechst, 33342 dyeing counting DNA.TRITC is meant with TRITC-link coupled second antibody and carries out mark (grating, 20 μ m).

Accompanying drawing 31 is that proof is compared with the parthenogenesis embryo, and AKAP95 is anchored on the figure in the protokaryon of nuclear transfer embryo more consumingly.This figure be presented at 3% Paraformaldehyde 96 fixing before, with 0.1%Triton X-100 and 1mg/ml DNA enzyme I and 100 or 300mM NaCl in-situ extraction after 30 minutes at room temperature, the lamin B, the AKAP95 that are not extracted of mark and the relative proportion of DNA in the protokaryon of parthenote (parthenotes), nuclear transfer embryo and somatocyte donorcells nuclear.Detect the location of Type B nuclear lamina (redness) and AKAP95 (green) with two immunofluorescences.Quantize the fluorescent mark intensity of each channel (channel)-redness (lamin), blue (DNA) and green (AKAP95).Reference value (100% is not extracted) be illustrated in fixing before with the relative content of painted Type B lamin, DNA and AKAP95 in the embryo of 0.1% saturatingization of Triton X-100 or the cell.Every group is detected 30 embryos approximately.

Accompanying drawing 32 is photos of the nuclear transplantation in cattle embryo of protokaryon phase, this embryo is merged and the activation of oocytes generation by inoblast, activate ovocyte 4 minutes with 5 μ M ionomycins, activate 4 hours with 10 μ g/ml cycloheximides/2.5 μ g/ml cytochalasin D then, as in (b '), activate with ionomycin/cycloheximide/cytochalasin D, (b ＂ CHX) or as (b ') cultivates (b ＂ ') with 10 μ g/ml radiating streptozotocin Ds in whole activation is handled then to cultivate 9 hours again with 10 μ g/ml cycloheximides.To resist lamin B (rabbit polyclonal) antibody and anti-Lamin A/C (mAb) antibody to be used for a kind of prepared product.Illustration is with the DNA photo of 0.1 μ g/ml Hoechst, 33342 marks (grating, 20 μ m).

Accompanying drawing 33 is that the chromatin in mitotic cell matter extract (M-S15), mitotic cell liquid extract (M-S200) and the ovocyte extract (MII-S15) concentrates and the figure (nucleus of n=300-400 detection in 3-5 is repeated) of nuclear envelope disintegration.

Accompanying drawing 34A-34C is the input ox inoblast nuclear (accompanying drawing 34A) of purifying and the number cover immunofluorescence analysis picture of the condensed chromatin in mitotic cell liquid extract (accompanying drawing 34B) and ovocyte extract (accompanying drawing 34C).Detect specified nuclear mark.Come dyeing counting DNA (grating, 10 μ m) with propidium iodide (redness).

Accompanying drawing 35 is in conventional nuclear transplantation (NT) or nuclear injection (NI) method and adopts method of the present invention chromatin to be expelled to the photo of a series of immunofluorescence analysis of the condensed chromatin that obtains behind the ovocyte (CT) in ovocyte.As if detectable lamin B and A/C are all dissolved (grating, 10 μ m).

Accompanying drawing 36A-36B is a series of immunofluorescence analysis photos by the protokaryon of chromatin transfer, nuclear transplantation or nuclear injection generation.Fixing and mark embryo when nuclear transplantation, nuclear injection or chromatin shift back 19 hours.Also detect the gynecogenic protokaryon (Part.) of contrast.Accompanying drawing 36A represents the analysis to Lamin A/C and B.Accompanying drawing 36B represents the analysis to AKAP95 and NuMA.Lamin A/C (Green Marker) only appears at nuclear transplantation and examines in the protokaryon of injecting (grating, 30 μ m).

Accompanying drawing 37 is the synoptic diagram of method that are used to produce clone's Tc calf.Show that the HAC construct has the hChr22 that contains Ig λ and IgH gene and the site of hChrl4 district and neo selective marker.From the CHO clone, HAC is transferred in the tire ox inoblast by the MMCT method.Tc inoblast and enucleation oocyte merged carry out nuclear transplantation.The Tc embryo of vitro culture reconstruct was implanted in the receptor cow then to blastula stage.Gestation is 60 days approximately, takes out the Tc fetus; Rebulid, estimate fibroblast, be used for further nuclear transplantation.The Tc embryo transfer of cloning is again produced the Tc calf in acceptor.

Accompanying drawing 38A-38D diagram is to the analysis of Tc fetus.G418 selects the Tc fibroblast of production again and the non-transgenic inoblast (accompanying drawing 38A) of contrast.IgH and Igl locus in genome PCRTc fetus and the contrast.Three fetus #5968 (swimming lane 1), #6032 (swimming lane 2) and #6045 (swimming lane 3) derive from Δ HAC inoblast, and fetus #5580 (swimming lane 4) is from Δ Δ HAC inoblast.In contrast, reclaim and estimate non-transgenic fetus (swimming lane N) (accompanying drawing 38B).In all Tc fetuses and positive control people liver dna sample (swimming lane P), all detect IgH and Igl locus by PCR, but in negative control, do not detect (swimming lane N).Reset and expressing human Ig μ and Ig λ transcript, these transcripts are by the spleen (swimming lane N) of RT-PCT from negative control non-transgenic ox, (the accompanying drawing 38C) that amplification obtains in people's spleen (swimming lane P) of brain (swimming lane 1), work (swimming lane 2) and spleen (swimming lane 3) and the positive control of clone Tc fetus.Reclaimed the people Ig μ that from clone Tc fetus, increases by RT-PCR and the representative Nucleotide and the deduced amino acid (accompanying drawing 38D) of Ig λ transcript at the 91st day.RT-PCR according to describe and carry out (Kuroiwa etc., Nature Biotech 18:1086-1090,2000).For people's Ig μ transcribed, VH1/5BACK, VH3BACK and VH4BACK were as 5 ' primer, and C μ-2 is as 3 ' primer.For people lg λ transcribed, V λ lLEA1, V λ 2MIX and V λ 3MIX were as 5 ' primer, and C λ MIX is as 3 ' primer.Clone the cDNA of test kit (Invitrogen) subclone amplification with TA, and check order with automatic sequencer (ABI3700 system).

The photo of accompanying drawing 39A-39C graphic analysis clone's Tc calf.Four-head Tc clones calf; From the male calves (#50) of clone #6045 with from the female calf (#1064, #1065, #1066) (accompanying drawing 39A) of clone #5968.Genome OCR is from IgH and the Ig λ locus of the PBLs of clone Tc calf and contrast; Calf #1064 (swimming lane 1), #1065 (swimming lane 2), #1066 (swimming lane 3), #50 (swimming lane 4), #1067 (swimming lane 5) and #1068 (swimming lane 6) (accompanying drawing 39B).In all Tc calves and positive control people liver dna (swimming lane P), detect IgH and Igl locus by genome PCR, but in the non-transgenic calf of negative control, do not detect (swimming lane N).The Metaphase Chromosome that fish analysis is dispersed in the cell shows single signal, and cell shows dual signal (accompanying drawing 39C).Arrow indication HAC is the location in the ox karyomit(e) around.As probe HAC is carried out paintedly with the people COT-1DNA of digoxigenin mark, detect with the rhodamine of anti-digoxigenin.

Accompanying drawing 40A-40B diagram lamin B, Lamin A/C, NuMA and the AKAP95 immunofluorescence distribution in the protokaryon phase of bovine fetal fibroblast (accompanying drawing 40A) and vitro culture and the ox embryo 8-16 cell stage (accompanying drawing 40B).NuMA and the AKAP95 mark of arrow points in female pronucleus (in two protokaryons less that).Illustration is the DNA with 0.1 μ g/ml Hoechst, 33342 marks.

Lamin B, Lamin A/C, NuMA and AKAP95 in accompanying drawing 40C diagram immunoassay ox inoblast (Fib) and the protokaryon embryo (PN).Grating, 10 μ m (accompanying drawing 40A), 50 μ m (accompanying drawing 40B).

The kinetics of nuclear envelope, NuMA and AKAP95 among the accompanying drawing 41A-41D diagram ox Nt embryo.With specific antibodies (TRITC), monitor fibroblastic nuclear decomposition of donor and the assembling (accompanying drawing 41A) of protokaryon newly at PCC and PN stage.Detect the parthenogenesis protokaryon (Part.PN) that generates with similar active program simultaneously.Provided percentage ratio (± SD) (n is about 20 embryo/groups, three repetitions) with the embryo of various antibody labelings.The acceptor ovocyte is activated 4 minutes with 5 μ M ionomycins, activate 4 hours (b) with 10 μ g/ml CHX/2.5 μ g/ml cytochalasin D then, as in (b), activate with ionomycin/CHX/ cytochalasin D, then cultivate 9 hours (b '), perhaps in whole activation processing, as in (b) and with 5 μ g/ml Act D, activate (b ＂) (accompanying drawing 41B) with 10 μ g/mlCHX.With DNA (illustration among the B) Hoechst 33342 marks.Grating, 10 μ m.Accompanying drawing 41C is NT embryo (100% fluorescence with respect to be untreated (-); N=30 embryo/group, three repetitions), the column diagram of average ± SD immune fluorescence intensity of the Lamin A/C among the NT embryo that CHX and ActD handle and B and NuMA.Accompanying drawing 41D is an associating column diagram in the nucleus of the AKAP95 in NT embryo's the protokaryon.Before fixing, extract parthenote (Part.) and the NT embryo (NT) and the donor inoblast (Fib) in PN stage with 0.1% Triton X-100/1mg/ml DNA enzyme I/300Mm NaCl.Detect lamin B and AKAP95 by immunofluorescence, and with Hoechst 33342 DNA that dye.Data are expressed as with respect to the fluorescence in the parthenogenesis embryo who is not extracted or NT embryo or the somatocyte (100% fluorescence, about 30 the embryo/groups of n, three repetitions), average ± SD fluorescent mark intensity of lamin B, DNA and AKAP95.Grating, 10 μ m.

Accompanying drawing 42A-42D proof CT produces the embryo who lacks Lamin A/C and NuMA.Detect the redistribution of monitoring Lamin A/C and B, NuMA and AKAP95 by in the fibroblastic nucleus of ox, carrying out immunofluorescence, the fibroblastic nucleus of this ox is cultivated (Chaudhary etc. in the fibroblastic extract of mitotic ox, J.Cell Biol.122:295-306,1993) (accompanying drawing 42A).With 0.1 μ g/ml propidium iodide marker DNA.Provide the dissociate nuclear ratio (n in three repetitions〉200 nucleus/mark) of (mark loses) of specific markers that shows as.Produce PN embryo and after activation 14 hours by CT, NT, NI or parthenogenesis, detect the distribution (Poccia etc. of Lamin A/C and B, NuMA and AKAP95 by immunofluorescence, Trends Biochem.Sci.17:223-227,1992 and Chaudhary etc., J.Cell Biol.122:295-306,1993) (accompanying drawing 42B).With Hoechst 33342 marker DNAs.Average ± SD the immune fluorescence intensity of specific markers is with respect to the fluorescence intensity among the NT embryo in PN stage (NT, 100% fluorescence in CT, NI or gynecogenic embryo; N=15-20 embryo/mark, three repetitions) (accompanying drawing 42C).After extracting with Triton X-100/DNA enzyme I/NaCl, lamin B, DNA among the NT in Pn stage and the CT embryo and the immune labeled intensity of AKAP95 (accompanying drawing 42D).Numeric representation is for respectively with respect to average ± SD fluorescence intensity (about 15 the embryo/mark/processing of n, three repetitions) of protokaryon phase NT that is not extracted and CT embryo.Grating, 10 μ m (accompanying drawing 42A), 20 μ m (accompanying drawing 42B).

Accompanying drawing 43A-43B illustrates external donorcells nuclear decomposition and the localized change of TBP in the protokaryon of the reconstruct that is produced by CT.In the condensed chromatin (NT PCC) in immunofluorescence analysis donor inoblast (Fib), PCC stage and mitotic division extract behind spissated donor karyomit(e) (MS 15 CC), NT protokaryon (NT PN) and the CT protokaryon (CT PN), the kinetics of TBP in NT and CT process relatively in accompanying drawing 43B.With Hoechst 33342 marker DNAs.Grating, 20 μ m.The TBP/AKAP95 in each stage and TBP/DNA fluorescence intensity are shown in accompanying drawing 43A and the gynecogenic protokaryon (Part.PN) (accompanying drawing 43B).Provide the average ± SD fluorescence intensity among every group of 10-13 embryo.

Accompanying drawing 44A is the synoptic diagram (pBPrP (H) KOneo carrier) that contains the prion protein knockout carrier of the present invention of neomycin resistance gene.How this schematic illustration identifies the clone of correct target practice.

Accompanying drawing 44B is synoptic diagram (pBPrP (H) KOpuro carrier) that contains the prion protein knockout carrier of the present invention of puromycin resistance gene and the method for identifying the correct clone who practices shooting.

Accompanying drawing 44C is listed in the different ox fibroblasts, the form of the homologous recombination frequency of prion protein knockout carrier of the present invention on the prion protein locus.

Accompanying drawing 45A diagram is from the photo of the level of lamin B, Lamin A/C, NuMA, AKAP95 and DNA in NT and SLOT embryo's the protokaryon.

Accompanying drawing 45B is the chart from the fluorescence intensity of accompanying drawing 45A.

Accompanying drawing 46A-46D is described in the fibroblastic nuclear kinetics behind the NT.Accompanying drawing 46A shows that lamin B, Lamin A/C and the TBP immunofluorescence in the ox inoblast and the ox embryo in IVP protokaryon (PN) and 8-16 stage distributes.Illustration is the DNA with Hoechst 33342 marks.Grating (left side) 10 μ m, (the right) 50 μ m.Accompanying drawing 46B is lamin B, Lamin A/C and the TBP immunoblotting picture in fetal fibroblast and protokaryon embryo.Accompanying drawing 46C is lamin B, Lamin A/C and the TBP immunofluorescence analysis in the PCC and the NT embryo in protokaryon (NT-PN) stage.Grating, 10 μ m.The column diagram of the immune labeled intensity that accompanying drawing 46D is a specified protein in IVP embryo's male (MPN) and female (FPN) protokaryon and NT embryo's protokaryon (NT-PN).Data are expressed as the average ± SD ratio of antibody fluorescence and Hoechst 33342 (DNA) fluorescence.(A, C, 3-5 D) repeat in to 20 above embryos of each labeled analysis.

The feature of the protokaryon among the accompanying drawing 47A-47C diagram NT embryo.Accompanying drawing 47A is after carrying out in-situ extraction with 1%Triton X-100/1mg/ml DNA enzyme I/300mM NaCl, and TBP and DNA (Hoechst 33342) are at protokaryon IVP and NT embryo and the immune labeled intensity in the inoblast (column diagram of mean number ± SD).Mark intensity is used with respect to the embryo's who is not extracted mark intensity and is represented (n=30 embryo or cell/group).Accompanying drawing 47B is the photo that is shown in the expression of mark among the NT embryo, and this embryo such as this paper describe (b) or be activated (n=30 embryo/group, 2-3 repetition) when having 10 μ g/ml CHX or (b ＂) 5 μ g/ml ActD.Illustration is DNA.Grating, 10 μ m.Accompanying drawing 47C is that (column diagram of mean number ± SD) and is compared (n=15-20 embryo/processing/mark) as the fluorescence of the Hoechst among the activated embryo among the accompanying drawing 47B 33342 (DNA) for the ratio of the immune labeled intensity of specified protein.

Accompanying drawing 48 diagram SLOT programs.Step 1 is reversibly changed donor inoblast 30 minutes thoroughly with 500ng/ml SLO.Step 2, the cell of processing is changed in washing thoroughly, and cultivates in containing the mitotic division extract of ATP regeneration system, makes karyomit(e) concentrate and promotion removal nuclear components (arrow).Step 3 is removed extract, randomly has 2mM CaCl ₂Substratum in closing cell 2 hours again.Step 4, with cytogamy in non-nucleus egg mother cell.Step 5 activates ovocyte as NT, make protokaryon form and growth.

Accompanying drawing 49A-49C is presented at and induces somatic nucleus disintegration in the mitotic division extract.Accompanying drawing 49A proof mitotic division extract can not cause apoptosis.Complete ox inoblast (swimming lane 1), with the inoblast (swimming lane 2) that 5 μ g/ml Luo Keda azoles are handled 20 hours apoptosis, perhaps will be exposed to the inoblast that mitotic division extract (swimming lane 3) or 1 hour SLO-of interval extract (swimming lane 4) change processing thoroughly and carry out immunoblotting (go up and organize) with anti--PARP antibody.In 0.8% agarose, carry out electrophoresis and estimate dna degradation with ethidium bromide staining.The inoblast that accompanying drawing 49B is changed processing thoroughly contain (+ATP) or not contain (after ATP) cultivating 45 minutes in the mitotic division extract of ATP-regeneration system, the picture of the immunofluorescence analysis of lamin B, Lamin A/C, TBP and AKAP95.Grating, 10 μ m.In 4 repetitions, more than 1000 analyzed cell demonstrates as directed stable labelling.Accompanying drawing 49C be immunoblotting assay interval inoblast (swimming lane 1), with 1 μ g/ml Luo Keda azoles turn to the inoblast (swimming lane 2) of m period synchronously, from being exposed to the mitotic division extract (swimming lane 3,4) that contains or do not contain the ATP-regeneration system or the quilt that is exposed to the interval extract changes into the isolating chromatin of fibrocyte thoroughly, and the whole picture that changes into fibrocyte (swimming lane 6) that is exposed to the mitotic division extract.Antibody with specific protein is detected spot.Histone H24 loads contrast as the albumen in the gel.

Accompanying drawing 50A-50D explanation SLOT embryo's nuclear feature.Accompanying drawing 50A diagram imports the morphology (arrow) in 30 minutes behind the ovocyte by SLOT or NT as described with chromatin.Dotted lines the tenuigenin of ovocyte.The one group of chromatinic enlarged view of donor that shows among SLOT and the NT embryo in the right.Grating, 20 μ m.Accompanying drawing 50B diagram lamin B, Lamin A/C and the TBP distribution in the embryo of protokaryon phase (last two groups) of producing and 8-16-cell stage (two groups down) by NT or SLOT.Grating, 20 μ m.(n=20-30 embryo/mark, three repetitions).Accompanying drawing 50C is for specified protein in NT or SLOT embryo's protokaryon, the ratio of immune labeled intensity and Hoechst33342 (DNA) fluorescence intensity (column diagram (about 20 embryos of n/mark, 3 repetitions) of mean number ± SD).Accompanying drawing 50D is with respect to the embryo who is not extracted, at the immune labeled intensity of extracting the TBP of back in protokaryon NT and SLOT embryo with 0.1% Triton X-100/1mg/ml DNA enzyme I/300mM NaCl (mean number ± SD) and the column diagram of DNA (Hoechst 33342).(n=15 embryo/group, 2 repetitions).

Accompanying drawing 51A-51E diagram is to SLOT and NT clone's growth and morphology evaluation.Accompanying drawing 51A is the column diagram of relevant NT and SLOT clone's ectogenetic ratio.Carry out ultrasound investigation at specific pregnant time and estimate pregnancy rate.Give calf natality and birth back (post-partum) calf surviving rate of 24 hours.After transplanting 59 SLOT embryos and 211 NT embryos, obtain 12 calves and 23 calves respectively, survive more than 8 weeks.Calculate the P value by the test of Fisher extract.Accompanying drawing 51B is the SLOT clone's (hybridization of Simintal * Angus is same female) in age in 5-6 week a photo.Accompanying drawing 51C is in birth 24 hours, the chart of the score value of single SLOT and NT calf and placenta.Accompanying drawing 51D is the chart of single SLOT and NT clone's birth weight.In accompanying drawing 51C and 51D, in SLOT (round dot) or NT (square) clone, a kind of color represents calf score value, placenta score value and the birth of same individuality heavy.Accompanying drawing 51E is the box-shaped pattern analysis (box plot analysis) of SLOT and NT clone's calf score value, placenta score value and birth weight.

Detailed Description Of The Invention

The present invention's part is based on adopting method described here with wonderful high-frequency deactivation bovine prion protein locus. A large amount of correct prions of practicing shooting knock out the production that cell is very easy to the ungulate (for example ox) that prion knocks out. In addition, the invention provides and improve improving one's methods of nuclear transfer efficient, knock out the production of ungulate with further simplification prion. The transgenic ungulates of these generations is expectation sources of agricultural product (for example meat) and medicine (for example by the people of one or more heterologous nucleic acids codings in the transgenic ungulates therapeutic antibodies).

Be used for being based in part at the knockout carrier of donor ox cell deactivation prion gene seat the sequence (AJ298878) of the bovine prion protein GFP seat among the Genbank that publishes. Be used for to make up knockout carrier the prion protein gene genome sequence the source with mated by the source of the fibroblast kind of genetic modification. Especially, the genome sequence of the prion protein gene of ox is used for making up the knockout carrier of effectively practicing shooting for the fibroblast of Niu Laiyuan. Knockout carrier contains two zones with prion gene seat homology, these two locus are continuous in the prion gene seat, and knockout carrier is designed to insert the drug resistant gene that tanscription termination (STOP) sequence and flank have two loxP sites in the prion gene seat. A kind of alternative scheme is to use two discontinuous homologous regions in knockout carrier, in order to delete two endogenous prion protein gene regions between the homologous region. In order to reduce the activity of prion protein, sequence is inserted into just the back of the initial ATG codon of exon 3. In order to carry out effective homologous recombination at locus, a homology zone is longer than other homology zone. Especially, the length in 3 ' district is 8.3kb, and is more much longer than 5 ' homologous region of 1.1kb. In order to concentrate the homologous recombination body, with negative selection marker for example the DT-A gene be connected on 5 short ' homologous region with the direction opposite with the prion protein gene. Before transfection, with suitable Restriction Enzyme linearisation knockout carrier, so that the pUC of basic plasmid vector such as pBluescript can be connected on the end of negative selection marker. In addition, before joining fibroblast, process linearizing knockout carrier with the HBS (Hepes buffer saline) that contains the 1mM spermidine, strengthen homologous recombination.

Polyamines and any other compound can be added in the targeting vector, to improve the efficient of homologous recombination. Preferred Concentrations of Polyamines is 0.1-10mM; Most preferably, concentration is 1mM. Polyamines can be selected from, for example, and spermidine, spermine, cadaverine and putrescine. Preferred polyamines is spermidine. Other compounds are selected from the compound that can form the ionic bond that connects DNA phosphoric acid residue, for example, and poly-l-lysine or poly L-arginine.

Be not exposed to the end of linearizing targeting vector by assigning negative selection marker to make it, improve homologous recombination efficiency. Preferably, 5 of the functional unit of negative selection marker ' and 3 ' end lay respectively at away from 5 of linearizing targeting vector ' and 3 ' terminal at least 1Kb part preferred 2Kb place at least. Usually, homologous gene seat sequence is positioned at 5 of negative selection marker ' and 3 ' end, so that the linearizing targeting vector of negative selection marker distance is terminal enough far. In linearizing targeting vector, the opposite side of negative selection marker is designed to keep at least additional sequences of 1Kb, preferred 2Kb. For example, the pUC carrier sequence in the targeting vector can be used as this additional sequences.

In a kind of ad hoc approach, the knockout carrier of Holstein Niu Laiyuan is transformed in the fetal fibroblast of Holstein ox by electricity, and acquisition semizygote target practice clone's frequency is higher than 50% astoundingly. A large amount of correct target practice clones are sufficient for effectively preparing healthy fetus and calf. In order to prepare fetus and the calf of isozygotying and practicing shooting, knock out fibroblast with the suddenly change remaining allele of prion gene seat of identical knockout carrier or knockout carrier electroporation with different antibiotics resistance genes with separating semizygote from semizygote target practice fetus. Alternately, knock out ox with female semizygote and produce the homozygous calf that knocks out by mating is male.

Produce the method for the ungulate of expressing xenoantibody

The importing of human immunoglobulin gene's seat is had the prion protein that isozygotys to be knocked out in the fibroblast of genotypic ox, knock out ox (prion protein KO/hAb ox) with the prion protein that isozygotys of producing people's antibody, comprise such as suitable method, the human artificial chromosome (HAC) that for example will contain heavy chain and light chain immunoglobulin gene is inserted in the ungulate or human B cell or B cell precursor (for example is inserted in foetal period or the postnatal ungulate, immune deficiency or immunosupress ungulate) (referring to, for example PCT discloses WO01/35735 and W002/074938).

Can the nucleic acid of expectation be inserted in the donorcells for the production of the transgenic ungulates of expressing xenoantibody with conventional method, contain the gene (preferred whole locus) of the antibody of producing specific species (for example people) in the gene of expectation. Preferably, use the human artificial chromosome, for example be disclosed among the open W097/07671 of PCT and the WOOO/10383 those. These human artificial chromosomes also are disclosed among the Japan Patent JP 30300092 that has authorized. Each piece of writing of these publications is incorporated herein by reference fully at this. In addition, human artificial chromosome or contain and the genetically modified construction method of expressing human immunoglobulin gene is described in Shen etc., Hum.Mol.Genet.6:1375-1382 (1997); Kuroiwa etc., Nature Biotechnol.18:1086-1090 (2000); Loupert etc., Chromosome 107:255-259 (1998); PCT open WO00/10383, W098/24893, W096/33735, W097/13852, W098/24884 and W097/07671; And in the United States Patent (USP) 5,877,397,5,874,299,5,814,318,5,789,650,5,770,429,5,661,016,5,633,425,5,625,126,5,569,825 and 5,545,806, each piece of writing is incorporated herein by reference fully at this. Can also the xenoantibody gene be imported in the wild type zooblast with the human artificial chromosome; This adopts said method to realize. By merging such as microcell described here, artificial chromosome is imported in the zooblast, particularly in the fetal fibroblast.

As using substituting of human artificial chromosome, can use will the encode nucleic acid of immunoglobulin (Ig) of yac vector, BAC carrier or cosmid vector to be incorporated in the chromosome. Adopt known method, comprise the yeast spheroplast fusion of yac vector etc. such as electroporation, fat transfection, usefulness, the carrier that will comprise xenogenesis Ig gene (is described in, for example PCT discloses W098/24893, W096/33735, W097/13852 and W098/24884, and United States Patent (USP) 5,849,992 and 5, in 827,690) import in the fetal fibroblast. In addition, the carrier that comprises xenogenesis Ig gene is practiced shooting on the endogenous Ig locus of fetal fibroblast, causes importing simultaneously xenogenesis Ig gene and destroys endogenous Ig gene.

Can also as the patent (with above) of Lonberg etc. described in, the integration of the nucleic acid of the heteroimmune globulin gene of implementing to encode. In " knocking in " construct for the chromosome that the heteroimmune globulin gene is inserted into the host ungulate, one or more immunoglobulin genes and antibiotics resistance gene are operably connected on the promoter, and this promoter is activated in the cell type of construct institute transfection. For example, form active, induction type or tissue-specific promoter is used to activate transcribing of integrated antibiotics resistance gene, so that can select transfected cell according to the antibiotic resistance that produces. Alternately, can use and knock in construct, the box of knocking in that wherein contains Ig gene and antibiotics resistance gene is not operatively connected on the promoter. In this case, based on the expression of the antibiotic resistance mark that under the regulation and control of internal promoter, produces, select to knock in the cell in the downstream of having integrated internal promoter in the box. These selected cells are used to nuclear transfer procedure described here, contain the transgenic ungulates of the heteroimmune globulin gene that is integrated in the host chromosome with production.

Adopt similar methodology, may prepare and insert to contain to be useful on and express for example gene of the Ig of dog, cat, other ungulates, inhuman primate wherein of different plant species. As mentioned above, also be known in the art, the immunoglobulin gene of species of the same race does not have substantial sequence homology between different plant species.

In case confirm, the artificial chromosome that is inserted into, for example the human artificial chromosome stably is inserted in the clone, and bovine fetal fibroblast for example is with the donor of this clone as nuclear transfer. This can determine by PCR method. The transgenosis calf that generates comprises the stable nucleic acid that imports, for example human artificial chromosome. Having obtained stable integration behind the calf of nucleic acid (for example, the human artificial chromosome), this animal is detected, determine whether they express people Ig gene immune and the affinity maturation response.

Method for reducing the production of the endogenous antibody in the ungulate of also expressing xenoantibody

Can also improve above-mentioned whole scheme. For example, before or after inserting heterologous nucleic acid, one or more endogenous of deactivation Ig gene in donorcells reduces the content of the expressed endogenous antibody of the transgenic ungulates of generation. In addition, the animal of xenogenesis Ig gene and the animal mating that endogenous Ig gene is inactivated will have been kept. The exemplary arrangement that is used for continued operation donorcells (for example ox becomes fibre property) is described hereinafter.

At first, produce the fetal fibroblast that the prion protein isozygoty knocks out. Then, produce the fibroblast that hemizygous cattle immune globulin heavy chain (BIgH) knocks out by gene targeting, produce fetus with method of nuclear transfer (for example SLOT or chromatin shift). Take turns gene targeting by second, produce the fibroblast that the BIgH that isozygotys knocks out, then produce fetus with method of nuclear transfer. Produce the fibroblast that hemizygous cattle immune globulin light chain (BIgL) knocks out by gene targeting, then produce fetus by nuclear transfer. Produce the fibroblast that the BIgL that isozygotys knocks out by the two-wheeled gene targeting, and produce fetus by nuclear transfer. Then, the human immunoglobulin gene seat is imported above-mentioned genotypic the knocking out continuously in the fibroblast that prion protein/BIgH/BIgL of isozygotying knocks out that have, produce fetus and calf by method of nuclear transfer.

Illustrative methods for the production of ungulate of the present invention

Be included in for the production of the method for optimizing of ungulate and the donor inhereditary material to be inserted into the acceptor egg mother cell to form this inhereditary material of reconstruct before the genetically modified ungulate, the ungulate that produces has sudden change and selectively has the nucleic acid of one and a plurality of coding xenoantibodies at endogenous prion nucleic acid. Reconstruct refers to the morphological change of the developmental potency of the nuclear transfer egg mother cell that any raising generates, and the nuclear transfer egg mother cell that this variation has improved gained is with respect to the growth of the egg mother cell of derivative by whole cell or complete nuclear transfer are entered the acceptor egg mother cell. Programming is by (for example programming culture medium again again, mitosis extract, detergent and/or salting liquid, perhaps protein kinase solution) cultivate in and in endogenous prion nucleic acid, have sudden change and have randomly that the nucleus of the nucleic acid of one or more coding xenoantibodies carries out, cause nuclear envelope to decompose and may cause chromatinic concentrated. This nuclear envelope decomposition and Chromatin condensation be so that the release of the albumen of transcriptional control and being attached on the chromosome, this will promote egg mother cell, embryo or development of fetus do not expect the genetic transcription wanted. Can before transferring to the acceptor egg mother cell at chromatin, be purified, to remove other modulin. Selectively, the specific modulin that discharges from chromosome can be consumed by immunity or remove from the culture medium of programming again (for example, cell extract), again is attached on the chromosome to prevent them. After nuclear transfer, form in the chromatin depolymerization with in egg mother cell in the process of nuclear envelope, may be incorporated on the chromosome from the cytoplasmic new albumen of egg mother cell. These protein promote genetic transcription, the offspring who allows Oocyte Development to become can survive.

The cloning process that another kind can be used for producing transgenic ungulates comprises by (for example programming culture medium again, cell extract) cultivate in thoroughly changed processing cell (namely, in endogenous prion nucleic acid, have sudden change and randomly have the cell of the nucleic acid of one or more coding xenoantibodies) added in this cell in the past or therefrom removed the factor, this cell of programming again. The plasma membrane of thoroughly being changed the cell of processing is preferably sealed to wrap up the factor of expectation again and is kept the cell membrane integrity. Then, with the cell transfer of again programming in the acceptor egg mother cell, to produce cloning mammal. This cloning process has been used to produce the fetus that does not contain heteroimmune globulin nucleic acid, and it was survived more than 60 days. Preliminary Results shows, adopts 40-60 days the fetal survival rate (7/10 of the fetus of this method production; 70%) is higher than conventional nuclear transfer fetus (8/16; 50%). Can expect in endogenous prion nucleic acid, to have the fetus that suddenlys change and/or have the nucleic acid of one or more coding xenoantibodies and also have similar result.

In a kind of alternative method, produce chimeric embryo, wherein the placenta tissue major part is from a kind of genetic origin, and fetal tissue's major part is from another kind of genetic origin. The less generation placenta deformity of this chimeric embryo, thus survival rate may be improved. In a kind of this method, from embryo in vitro fertilization or natural embryo's cell with from having sudden change in the endogenous prion nucleic acid and randomly having embryo's the cells contacting of the nucleic acid of one or more coding xenoantibodies, described embryo produces with conventional method of nuclear transfer or any other cloning process described here. For example, can be injected into the periphery (for example, between oolemma and embryo itself) of nuclear transfer embryo from embryo's in vitro fertilization cell. This method is for the production of the chimeric embryo that does not contain the xenoantibody gene, and with 25% survival rate of contrast nuclear transfer embryo, it had 67% survival rate and compares in the time of 40 days. Can expect also have similar result from the cell of nuclear transfer embryo, the embryo of this nuclear transfer has sudden change and/or has the nucleic acid of one or more coding xenoantibodies in endogenous prion nucleic acid. In a kind of alternative method, allowing mutually to identify to produce under the condition of single chimeric embryo from each embryo's cell, use from the cell of the nuclear transfer embryo before fine and close and cultivate from the IVF Embryos before fine and close or natural embryo's cell (Wells and Powell, Cloning 2:9-22,2000). In these two kinds of methods, preferentially be attached in the placenta from embryo in vitro fertilization or natural embryo's cell, and preferentially be attached in the fetal tissue from the cell of method of nuclear transfer.

State of the art before the present invention

Although expressed people's Ig in mouse, because the difference of gene structure, antibody generting machanism and the B cell function aspect of antibody, whether people's Ig can trickle rearrangement and expression occur in ox or other ungulates can not be expected. Especially, different from mouse, the immunological recognition of ox and sheep and human different (Lucier etc., J.Immunol.161:5438,1998; Parng etc., J.Immunol.157:5478,1996; And Butler, Rev.Sci.Tech. 17:43,2000). For example, compare with the gene rearrangement in the mouse with human, the antibody gene diversity in ox and the sheep depends on the gene conversion more. In addition, in human and mouse, the B cell is positioned in the marrow at first, and in ox and sheep, the B celluar localization is in ileum Pai Er aggregate nodules. Therefore, before the present invention, may be difficult to, be not impossible, and the expection immunoglobulin (Ig) is reset and whether the variation of human immunoglobulin gene's seat occurs in the B clone of ox (perhaps other ungulates). In addition, also be difficult to be expected at when lacking its endogenous Ig or when being subjected to people's the interference of antibody, whether ox can survive, and namely produces normal immunologic function. For example, still whether the ox B cell of uncertain expression people Ig can correctly move in the ileum Pai Er aggregate nodules in the ox body, and this is impossible in human body. In addition, do not know whether normal people's Fc function of receptors is in the ox system, the receptor-mediated complement activation of Fc, the inducing cell factor discharge and antigen is removed. Because whether uncertain people's Ig will functionally be expressed, and is perhaps expressed to produce enough immune responses with q.s, therefore is difficult to expect whether this ungulate can survive. In addition, whether uncertain human chromosomal is stably maintained in the transgenic ungulates.

Be used for of the present invention method though described in the above, all technology will be illustrated in greater detail hereinafter.These embodiment are provided to illustrate the present invention, and are not interpreted as limiting the present invention.Especially, though these embodiment concentrate on transgenic cattle, described method can be used for producing and testing any transgenic ungulates.

Embodiment 1: the transgenic ungulates that prion protein activity reduces

Produce the ox fibroblast cloning that two allelotrope of PrP locus are wherein all suddenlyd change by the homologous recombination of order.By inserting Xin Meisu or tetracycline gene (neo or puro behind the initial ATG codon at exon 3 just, as described in Example 3) and Transcription Termination box (STOP), will be used to produce the PrP DNA construct that knocks out cell of isozygotying and be used for preventing transcribing of functional total length PrP mRNA.Therefore, the immature PrP transcript of generation lacks functional coding region.At first, DNA construct (the being the PrPKOneo carrier) electroporation that will contain the neo gene separates the neomycin resistance clone then in the ox fibroblast.According to pcr analysis, in some clones, homologous recombination takes place in exon 3.Therefore, generated the ox inoblast that an allelotrope in the PrP locus is suddenlyd change.(half-PrPKO) the inoblast, produced wherein a fetus that allelotrope is suddenlyd change in three PrP locus, and rebulid hemizygous fibroblast from the sudden change of partly isozygotying.Then, the DNA construct (being the PrPKOpuro carrier) that another kind is contained the puro gene for the second time electroporation in hemizygote-PrPKO inoblast, separate the tetracycline resistance clone then.According to pcr analysis, in some clones, in remaining allelic exon 3 with about 5% ratio generation homologous recombination.In this way, produced the ox fibroblast cloning (PrPKO isozygotys) that two allelotrope in the PrP locus are wherein all suddenlyd change.

From (isozygoty-produce 10 fetuses PrPKO) the inoblast, wherein two of the PrP locus allelotrope are all suddenlyd change, and rebulid and isozygoty-the PrPKO fibroblast by homeotic mutation.These isozygoty-the PrPKO inoblast in, analyze to determine to lack fully PrP by RT-PCR and express.Can also knock out cell production and isozygoty-the PrPKO inoblast by selecting to isozygoty in conjunction with same knockout carrier with the higher concentration microbiotic.By nuclear transplantation, from isozygoty-the PrPKO inoblast produces and to isozygoty-the PrPKO calf.

These methods are further described hereinafter.

The structure of prion protein knockout carrier

Following production prion protein knockout carrier (accompanying drawing 44A).For the locus DNA around the exon 3 that separates the prion protein gene, with following primer to coming the pcr amplified dna probe: 5 '-CCA CAT AGG CAG TTG GAT CC-3 ' (SEQ ID NO:69) and5 '-ATA AGA GGC CTG CTC ATG GC-3 ' (SEQ ID NO:70).Use this probe, screen ox (Holstein) locus lambda particles phage library, determine the positive colony of four lambda particles phages.With the further clone who analyzes among four clones of restricted mapping.The segmental 8.3kb BamHI of BamHI-BglII locus fragment (3 ' homology arm) subclone that will contain exon 3 and 1.2kb is in pBluescriptIISK (-), then neo and STOP expression cassette are inserted on the Bam HI site, this site is just in time in initial ATG codon back.The BglII site is changed into flush end, in this site, insert the NotI-joint.(DT-A Gibco) adds on the newly-generated NotI site with the diphtheria toxin gene then.In the target practice box, insert DT-A with the direction opposite, to kill the box of wherein practicing shooting by the cell (pBPrP (H) Koneo carrier) of random integration in the genome with neomycin resistance gene.Also made up similar knockout carrier (pBPrP (H) the Kopuro carrier that contains the puro gene; Accompanying drawing 44B).Can come these prion protein knockout carriers of linearizing with SrfI Restriction Enzyme (TOYOBO), pBluescriptIISK (-) is connected on the DT-A gene.

Transfection/knock out program

With following conventional electroporation scheme, carry out the transfection that male Holstein fetal fibroblast is.The solution that is used to cultivate bovine fetal fibroblast contain 500ml α MEM (Gibco, 12561-049), 50ml foetal calf serum (Hy-Clone # ABL13080), 5ml penicillin-Streptomycin sulphate (Sigma) and 1ml 2 mercapto ethanol (Gibco/BRL # 21985-023).In the day before yesterday of transfection, cell is implanted in the T175 tissue culture flasks, reach the rate of converging of 80-100%, determine by microscopy.On the same day of transfection, with about 10 ⁷Individual ox inoblast trypsinized, and with α MEM solution washing once.Behind the re-suspended cell, 30 μ g pBprion protein (H) the KOneo carriers that are dissolved among the HBS (Hepes buffer saline) that contains the 1mM spermidine are added in the cell suspending liquid in 800 μ l α MEM, pressure-vaccum is with thorough mixing repeatedly.Cell-DNA suspension is transferred in the electroporation test tube, under 550V and 50 μ F, carried out electroporation.After this, will be added on 60 24-hole flat boards by the cell of electroporation with the α MEM solution that has added serum.Cultivate after 48 hours, with the substratum replacement medium of the G418 that contains 500 μ g/ml, culturing cell 2-3 week, to select the neomycin resistance cell.After the screening, all clones that picking nearly 100% converges also are distributed in two flat boards (24-hole flat board).Cell in flat board is used for the locus DNA extraction, and the cell in another flat board is used for nuclear transplantation.From the clone, extract genomic dna, by the homologous recombination incident of PCR screening expectation.Typically, analyze about 100 G418-clone.

Screening is practiced shooting and is integrated

As mentioned above, adopt PUREGENE DNA separating kit (Gentra Systems),, from each 24-hole, extract genomic dna independently according to manufacturer's scheme.Each genomic dna is resuspended among 20 μ l 10mM Tris-Cl (pH8.0) and the 1mM EDTA (EDTA).Use following primer to screening by PCR: F7 (5 '-TGT CAAAGA GAC ACT CCT CAT TTG TCT TCC-3 ', SEQ ID NO:71) and R7 (5 '-TCA TAG CCG AAT AGC CTC TCC ACC C-3 ', SEQ ID NO:72).The sequence of a primer is arranged in knockout carrier, and the sequence of another primer is arranged in the outside (accompanying drawing 44A) of the integrative vector of the endogenous gene seat of being practiced shooting just.Therefore, in the time of only when knockout carrier is integrated into by the target practice locus by homologous recombination in, just can detect the PCR product of expectation.The PCR reaction mixture contains 17.9 μ l water, 3 μ l 10X LA PCR damping fluid II (Mg ²⁺Add), 4.8 μ ldNTP mixtures, 10pmol forward primer, 10pmol reverse primer, 2 μ l genomic dnas and 0.3 μ lLA Taq.By incubation reaction mixture under the following conditions, carry out 30 PCR circulations: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ of 10 seconds and 68 ℃ 5 minutes.Behind the PCR, by the electrophoretic analysis reaction mixture.From 94 Xin Meisu quick clones that screened of about 50%, generate the PCR product (accompanying drawing 44B) of expectation size. also by the another kind of primer of pcr amplification to confirm these positive colonies: F10 (5 '-TGA AGA TGT CCC AGT GCT GGG GAT-3 ', SEQ ID NO:73) and R10 (5 '-GAG TTA GGG GCG GGA CTA TGG TTG C-3 ', SEQ IDNO:74), generate the PCR product of the expection size of 2.7kb.In another fibroblast, female F1 (Holstein x Jersey) clone, also reproduced and surpassed 50% high homologous recombination frequency (accompanying drawing 44C).Therefore, no matter employed initiator cell system be what, successfully produced the ox fibroblast that an allelotrope of prion gene seat is wherein suddenlyd change by knockout carrier.

Chromatin shifts

The hemizygote Protein virus knocks out cell or can be used in any nuclear transplantation method described here from the nucleus of these cells, to produce the ungulate (calf that for example, knocks out) that the hemizygote Protein virus knocks out.If desired, can use as follows from the cell of the hoof class animal that knocks out fetus or live and produce the Protein virus of isozygotying and knock out cell.In a kind of ad hoc approach, be processed into fibrocyte (for example Niu Chusheng fetal fibroblast) 18 hours with 1 μ g/ml Luo Keda azoles, make it in the interim synchronization of mitotic division, the method (shake-off) of shaking off by m period is collected, washed twice in the physiological saline of phosphoric acid buffer, with at cytolysis damping fluid (20mM Hepes, pH8.2,5mM MgC1 ₂, 10mM EDTA, 1mMDTT and proteinase inhibitor) and middle washing is once.Sedimentary cell is resuspended in the long-pending ice-cold cytolysis damping fluid of monoploid, expanded 1 hour, carry out DounceShi homogenate with suitable glass pestle on ice.Under 4 ℃, with 15, the centrifugal lysate of 000x g 15 minutes, five equilibrium supernatant liquor (mitotic division extract), freezing in liquid nitrogen, be kept under-80 ℃.Use fresh or the refrigerated extract.

With 20hpm, with the ovocyte stoning of maturation in vitro.Will be from selected clone's transfected bovine fetal fibroblast with not containing Ca ²⁺/ Mg ²⁺HankShi balanced salt solution (HBSS) washing, in about 37 ℃ water-bath, adding 31.2U streptolysin O (SLO; Sigma) culturing cell is changed thoroughly in the suspension of 100 μ lHBSS.Absorb impervious DNA dyestuff iodate third key (0.1 μ g/ml) evaluation by film.With inoblast precipitation, the washing of changing thoroughly, and under about 37 ℃, contain in the mitotic division extract (1mM ATP, 10mM phosphocreatine and 25 μ g/ml sarcosine kinases) of ATP-generation structure incubation 30-45 minute at 40 μ l.With 0.1 μ g/ml Hoechst, 33342 mark aliquots containigs, monitoring chromatin concentrates.Behind the incubation, with 500 μ l α MEM/10% foetal calf serum (Hyclone) diluted reaction mixtures.With cytogamy in non-nucleus egg mother cell; Activate ovocyte with 28hpm, extracorporeal culturing embryo is to blastula stage.Every female receptor is transplanted two pieces of embryos.Monitor gestation by ultrasound examination, acceptor is carried out C-cut into slices and reclaim fetus with production clone.

Second takes turns transfection/knock out program

The following transfection of carrying out hemizygote-PrPKO fetal fibroblast system, wherein first KO carrier is integrated on " allelotrope A ".The solution that is used to cultivate bovine fetal fibroblast contain 500ml α MEM (Gibco, 12561-049), 50ml foetal calf serum (Hy-Clone # ABL13080), 5ml penicillin-Streptomycin sulphate (Sigma) and 1ml 2 mercapto ethanol (Gibco/BRL # 21985-023).In the day before yesterday of transfection, cell is implanted in the T175 tissue culture flasks, reach the rate of converging of 80-100%, determine by microscopy.On the same day of transfection, with about 10 ⁷Individual ox inoblast trypsinized, and with α MEM solution washing once.Behind the re-suspended cell, the 30 μ g KO carriers { pBPrP (H) KOpuro carrier } that are dissolved among the HBS (Hepes buffer saline) that contains the 1mM spermidine are added in the cell suspending liquid in 800 μ l α MEM, pressure-vaccum is with thorough mixing repeatedly.Cell DNA suspension is transferred in the electroporation test tube, under 550V and 50 μ F, carried out electroporation.After this, will be added on 30 48-hole flat boards by the cell of electroporation with the α MEM solution that has added serum.Cultivate after 48 hours, with the substratum replacement medium of the tetracycline that contains 1 μ g/ml, culturing cell 2-3 week, to select the tetracycline resistant cell.After the screening, all clones that picking nearly 100% converges also are distributed in two flat boards (24-hole and 48-hole flat board).Cell in flat board is used for the locus DNA extraction, and the cell in another flat board is used for nuclear transplantation.From the clone, extract genomic dna, by the homologous recombination incident of PCR screening expectation.

Be used for the screening that homology is practiced shooting and integrated

As mentioned above, adopt PUREGENE DNA separating kit (Gentra Systems),, from each 24-hole, extract genomic dna independently according to manufacturer's scheme.Each genomic dna is resuspended among 20 μ l10mM Tris-Cl (pH8.0) and the 1mM EDTA (EDTA).Use following primer to screening: F14 by PCR; 5 '-TTG CTCCAC CTT CCG TCT TCT GTC-3 ' (SEQ ID NO: 97) and R14; 5 '-GTTGGC GCC TAC CGG TGG ATG TG-3 ' (SEQ ID NO: 98).The sequence of a primer is arranged in the KO carrier, and the sequence of another primer is being positioned at the outside (accompanying drawing 1) that is integrated carrier just by in the endogenous gene seat of practicing shooting.Therefore, in the time of only when the KO carrier is integrated into by the target practice locus by homologous recombination in, just can detect the PCR product of expectation.The PCR reaction mixture contains 17.9 μ l water, 3 μ l 10X LA PCR damping fluid II (Mg ²⁺Add), 4.8 μ l dNTP mixtures, 10pmol forward primer, 10pmol reverse primer, 2 μ l genomic dnas and 0.3 μ lLA Taq.By incubation reaction mixture under the following conditions, carry out 30 PCR circulations: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ of 10 seconds and 68 ℃ 5 minutes.Behind the PCR, by the electrophoretic analysis reaction mixture.Screen 332 clones, 15 clones (#26,86,113,116,118,173,177,213,235,250,251,266,285,318,330) have the PCR product of expectation as a result, confirm by order-checking PCR product.

Based on polymorphic sequence, in all clones, the KO carrier is integrated among " the allelotrope B " of exon 3 of PrP locus.8 clones are used for nuclear transplantation and produce fetus, and from 3 fetuses (#1395,1468,1476) of #285 with from 7 fetus (#1439-1 of #113,1439-2,1487,3583,3632,3682-1,3682-2,4961) be proved to be the PrP KO that isozygotys, wherein puroKO carrier and neoKO carrier are integrated into respectively in " allelotrope B " and " allelotrope A ".Therefore, successfully produced 10 ox fibroblasts, wherein two of the PrP locus allelotrope are all suddenlyd change by the KO carrier.

Table 1 has been summed up and the PA data of carrying out with the PrP KO clone of isozygotying who derives from hemizygote PrP KO clone after chromatin shifts.

Table 1

The checking of the expression of PrP of isozygotying-lacking in the PrP fetus

From some fetuses, set up inoblast, and extracted total DNA with RNeasy Mini test kit (QIAGEN).The total DNA of 1 μ l is carried out the first chain cDNA analyze (the subscript first link analysis system (SuperScript First-Strand SynthesisSystem for RT-PCR) that is used for RT-PCR; Invitrogen), then carry out RT-PCR.According to the following RT-PCR that carries out.Used primer is 5 '-AAG AAG CGA CCA AAA CCT GG-3 ' (SEWQ ID NO:75)With 5 '-GTA ACG GTG CAT GTT TTC ACG-3 ' (SEQ ID NO:76)The PCR reaction mixture contains 32.5 μ l water, 5 μ l 10X Ex Taq damping fluids (TAKARA), 8 μ l dNTP mixtures, 10pmol forward primer, 10pmol reverse primer, 2 μ l, the first chain cDNA and 0.5 μ l Ex Taq (TAKARA).By incubation reaction mixture under the following conditions, carry out 35 PCR circulations: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ 10 seconds, 60 ℃ of 30 second and 72 ℃ 1 minute.Behind the PCR, by the electrophoretic analysis reaction mixture.Though in wild-type cell and hemizygote PrP KO cell, all detect PCR product clearly, 10 kinds isozygoty-all do not have positive PCR product in PrP KO clone any.This result proves, isozygoty-PrP KO clone lacks PrP fully and expresses.

The importing of embodiment 2:HAC and rearrangement

Be used to insert the summary of the method for HAC

For in one or two allelotrope of prion gene, having sudden change and expressing the production of the ungulates equipotential (for example ox) of heteroantibody, can adopt ordinary method that the nucleic acid (for example HAC) of expressing heteroantibody is inserted in the cell.For example, insert HAC before or after can being inactivated at one or two allelotrope of prion gene.If desired, the one or more endogenous antibody genes in the cell are also suddenlyd change.Then, this cell is used for conventional nuclear transplantation method,, carries out more detailed description hereinafter to produce the transgenic ungulates of expectation.

In fact, the male and female bovine fetal fibroblast that has obtained to contain human artificial chromosome's (for example, #14fg., #2fg., and #22fg.) is, and is produced clone's calf by these clones.

For example, insert simultaneously or in turn and derive from human chromosomal #14 (" #14fg " comprises the Ig heavy chain gene), human chromosomal #2 (" #2fg " comprises Ig κ chain gene and the human chromosomal #22 HAC of (" #22fg " comprises Ig λ chain gene).

Estimate by male #14fg animal and female #2fg and #22fg animal mating and to the offspring, to detect the transmission of these chromosome segments.If it is successful transmitting, two strains of mating contain the clone of all three chromosome segments with production so.

In addition, #14fg., #2fg. and #22fg. chromosome segment being inserted in the fetal cell (for example genetically modified H/L of isozygotying fetal cell) produces clone's calf or transgenosis HAC calf and other transgenosis calves (the H/L calf of for example isozygotying) is hybridized.Alternately, as mentioned below or adopt any other chromosome transfer method, insert other HAC, for example Δ HAC or Δ Δ HAC.

Ultimate principle

It is useful that the kind system of HAC transmits for HAC being imported animal (for example Ig knock-out animal), and is used to breed in groups animal.The key of transmitting HAC by strain is chromosomal material incomplete pairing in the reduction division process.But, prove that as (Proc.Natl.Acad.Sci.USA, 97:722,2000) such as Tomizuka successfully having carried out kind is transmission in mouse.

Method described in the accompanying drawing 1A comprises #14fg. is inserted in the male sex cell system, and #2fg. and #22fg. are inserted into respectively in the female cell system.Generation has the calf of HAC, kind is transmission by female and male can the detection.Contain heavy chain and light chain HAC among offspring's (about 25%) that part generates.Further hybridization generates the calf strain that contains all three kinds of chromosome segments.Selectively, these animals are used for and transgenic animal (the H/L animal of for example isozygotying) hybridization, and as described previously, these transgenic animal are by fetal cell production.

Test design

Cell obtains from initial cell line selection.These cells are Holstein or the clone that is different from top used those cells.This makes can hybridize and keep genetic diversity as much as possible in colony.Then with in the HAC transfered cell and select positive cell line.Selected clone is used to nuclear transplantation and produces calf.Since 12 monthly ages, collect seminal fluid and ovum, fertilization, and be transplanted in the receptor.Cell sample is used for dna marker analysis and karyotyping.From birth, sample of blood, drawn, and analyze the Ig albumen that whether has the people.

As noted before, adopt the method for above-mentioned developing test, also can randomly HAC be transferred in several clones of the H that isozygotys.

Provide the following examples to come further illustration the present invention extraly.

Insert the illustrative methods of HAC

Carry out other and test and prove, can produce xenogenesis (for example people) heavy chain immunoglobulin (μ) and lambda light chain by the ox host individually or in combination.In addition, these evidences, the human normal immunoglobulin chain is rearranged, and obtains polyclonal serum.In these methods, the gene that adopts the human artificial chromosome will express immunoglobulin (Ig) is inserted in the ox inoblast.Then, inoblast is used to nuclear transplantation, obtains fetus, and analyzes antibody producing.These methods and result will be described hereinafter in further detail.Expection can obtain similar result from inoblast, separate these inoblasts and they are used for the method for importing HAC described below from the fetus that has sudden change one or two allelotrope of prion gene.Alternately, in the Hac transfered cell, (i) imports sudden change or (ii) imports sudden change with the cell with HAC in from the cell of the clone fetus that is generated with HAC in the prion gene of cell then.

The HAC construct

With previously described karyomit(e) cloning system (Kuroiwa etc., Nature Biotech.18:1086-1090,2000), make up human artificial chromosome (HAC).In brief, structure for Δ HAC, cut off (Kuroiwa etc. by the directed karyomit(e) of telomere, Nucleic AcidRes., 26:3447-3448,1998), in human chromosomal 22 fragments (hChr22) of cutting off the loxP sequence that on the HCF2 locus, contains integration of previous report on the AP000344 locus.Then, by merge cut off at AP000344 locus place contain above-mentioned hChr22 fragment (hCF22) but DT40 cell clone and the DT40 cell clone (being called " R clone ") that contains the minimum karyomit(e) SC20 of the people carrier that stable strain transmits, founder cell hybrid.On the segmental RNR2 locus of S20, insert the loxP sequence and produce the SC20 carrier.The SC20 fragment is the natural fragment that derives from human chromosomal 14, comprises the whole zone (Tomizuka etc., Proc.Natl.Acad.Sci.USA 97:722,2000) of human Ig heavy chain gene.The DT40 cell hybrid that generates contains two kinds of hChr fragments.With Cre recombinase expression vector transfection DT40 hybrid, to induce the chromosome translocation of between hCF22 and SC20 carrier, carrying out the Cre/loxP mediation.With the stable transfectant of nested pcr analysis, to determine to have cloned the hChr22 zone of 2.5 megabasses on the loxP of SC20 carrier cloning site, this zone is limited by HCF2 and AP000344 locus.Then, by the FACS cell divide,, separate the PCR positive cell that expectation contains Δ HAC according to the fluorescence of coded green fluorescent protein.The cell of classification is carried out fluorescence in situ hybridization (FISH) analyze, contain the existing of Δ HAC of 2.5 megabasse hChr22 insets with checking.

Similarly, also adopt the karyomit(e) cloning system to make up Δ Δ HAC.Cut off the hChr22 fragment at AP000344 locus place, the loxP sequence is incorporated on the AP000553 locus in the DT40 cell by homologous recombination then.Then, the cell of generation merges with the R clone who contains the minimum chromosome vector of SC20.With Cre-expression vector transfectional cell hybrid, to carry out the chromosome translocation of Cre/loxP mediation.Determine Δ Δ HAC by PCR and fish analysis, it contains by the hChr22 inset of 2.5 megabasses of AP000553 and the qualification of AP000344 locus.

The gomphosis mouse that contains HAC by generation is estimated in the body of Δ HAC and Δ Δ HAC functional.Adopt ordinary method (the Japanese Patent 2001-142371 of application on May 11 calendar year 2001) then, these HAC are imported to respectively in mouse embryonic stem (ES) cell, produce gomphosis mouse with these stem cells.The mouse that generates has high mosaic (85-100% fur color), proves that the ES cell that contains these HAC has the interior mitotic division stability of body of high-caliber versatility and these HAC.In addition, Δ HAC is delivered to the next generation by Δ HAC gomphosis mouse strain, has proved the maiotic stability of this Δ HAC.

The chicken DT40 cell that has kept these HAC is according to budapest treaty, submit to Independent Administrative Leged Industrial Technology Complex Inst to specially permit biological sustenance center (International Patent organism Depository May 9 calendar year 2001, National Institute ofAdvanced Industrial Science and Technology, Tsukuba Central 6,1-1, Higashi1-Chome Tsukuba-shi, Ibaraki-ken, 305-8566 Japan) preservation.Preserving number is as follows: Δ HAC (FERM BP-7582), Δ Δ HAC (FERM BP-7581) and SC20 fragment (FERM BP-7583).The chicken DT40 cell that keeps these HAC also has been deposited in food Industry in Taiwan research and development institute (Food Industry Research andDevelopment Institute) (FIRDI).Preserving number and preservation date are as follows: (CCRC 960144 for Δ HAC; November 9 calendar year 2001), (CCRC 960145 for Δ Δ HAC; November 9 calendar year 2001) and the SC20 fragment (this clone is submitted preservation to title SC20 (D); CCRC 960099; On August 18th, 1999).

The hChr22 of 2.5 megabasses (Mb) among the Δ HAC inserts fragment and is made of following BAC contig, and described contig is listed by following Genbank accession number: AC002470, AC002472, AP000550, AP000551, AP000552, AP000556, AP000557, AP000558, AP000553, AP000554, AP000555, D86995, D87019, D87012, D88268, D86993, D87004, D87022, D88271, D88269, D87000, D86996, D86989, D88270, D87003, D87018, D87016, D86999, D87010, D87009, D87011, D87013, D87014, D86991, D87002, D87006, D86994, D87007, D87015, D86998, D87021, D87024, D87020, D87023, D87017, AP000360, AP00361, AP000362, AC000029, AC000102, U07000, AP000343 and AP000344.The hChr22 of 1.5Mb among the A Δ HAC inserts fragment and is made of following BAC contig: AP000553, AP000554, AP000555, D86995, D87019, D87012, D88268, D86993, D87004, D87022, D88271, D88269, D87000, D86996, D86989, D88270, D87003, D87018, D87016, D86999, D87010, D87009, D87011, D87013, D87014, D86991, D87002, D87006, D86994, D87007, D87015, D86998, D87021, D87024, D87020, D87023, D87017, AP000360, AP00361, AP000362, AC000029, AC000102, U07000, AP000343 and AP000344 (Dunham etc., Nature 402:489-499,1999).

The generation of bovine fetal fibroblast

In order to produce bovine fetal fibroblast, collect 45-60 days fetus the Holstein cow of the process disease examination of in the tame animal house of Trans Ova (Iowa), raising or the Jersey cow, wherein write down male parent and maternal triple-substituted pedigree continuously.With collected fetus at the wet Hematech ' s Worcester Molecular Biology Division that is transported on ice, to produce former generation fetal fibroblast.After the arrival, fetus is transferred in the 100mm plastic culture dish of non-tissue culture level of tissue culture cabinet.Use aseptic nipper and scissors, from described fetus, remove extraembryonic membrane and umbilical cord.After fetus transferred to new plastic culture dish, remove head, limbs and internal organ.The fetus that gills is transferred in the 3rd culture dish that contains the 10ml fetus washing fluid of having an appointment, and described fetus washing fluid is made up of following material: 125ml contains Ca ²⁺And Mg ²⁺1 * Dulbecco ' s-PBS (D-PBS) (Gibco-BRL, catalog number (Cat.No.) 14040); 0.5ml tylosin tartrate (Tylosine Tartrate) (8mg/ml, Sigma, catalog number (Cat.No.) T-3397); 2ml penicillin-Streptomycin sulphate (Sigma, catalog number (Cat.No.) P-3539); With 1ml amphotericin (Fungizone) (Gibco-BRL, catalog number (Cat.No.) 15295-017) (mixing is also passed through 0.2 μ m nylon filtration unit [Nalgene, catalog number (Cat.No.) 150-0020] and is filtered).

With the fetus washing fluid each fetus is washed 3 times again,, it is transferred in the 50ml taper tissue culture tube, it is chopped into small pieces lightly with the aseptic operation blade to remove micro blood.With no Ca ²⁺And Mg ²⁺1 * D-PBS (Gibco-BRL, cat#.14190) washing tissue once.After tissue is deposited to the pipe bottom, remove supernatant liquor, replace cell dissociation damping fluid (Gibco-BRL, catalog number (Cat.No.) 13151-014) into about 30ml.To manage and be inverted for several times, so that allow its mixing, then in incubator for tissue culture, at 38.5 ℃/5%CO ₂Following incubation 20 minutes.After tissue is deposited to the pipe bottom, remove supernatant liquor, be replaced with isopyknic fresh cell dissociation damping fluid.Tissue and cell dissociation buffer solution mixture are transferred in the flask of aseptic glass trypsinized of a 75ml who has a 24mm nose circle rotating rod (WheatonScience Products, cat #.355393).Flask is transferred to 38.5 ℃/5% CO ₂In the incubator for tissue culture, place on the magnetic agitation plate, stirred about 20 minutes to be enough to effective blended speed.Flask is transferred in the tissue culture cabinet; Make the tissue sedimentation, remove supernatant liquor then, by with 1,200rpm collected dissociated cell in centrifugal 5 minutes.Cell precipitation is resuspended in fibroblastic perfect medium of small volume this substratum composed as follows: 440ml α MEM (BioWhittaker, catalog number (Cat.No.) 12-169F); 50ml is through the foetal calf serum of irradiation; 5mlGLUTAMAX-I additive (Gibco-BRL, catalog number (Cat.No.) 25050-061); 5ml penicillin-Streptomycin sulphate (Sigma, catalog number (Cat.No.) P-3539); 1.4ml2-mercaptoethanol (Gibco-BRL, catalog number (Cat.No.) 21985-023) (all components except foetal calf serum is mixed, filter by 0.2 μ m nylon filtration unit [Nalgene, catalog number (Cat.No.) 151-4020] then), and storing on ice.Repeat described dissociation process 3 times again, in each step, use other 30ml cell dissociation buffer.Cell is merged; Wash with the inoblast perfect medium; Order at last by 70 μ m cell filters (B-D Falcon, catalog number (Cat.No.) 352350), produces single-cell suspension liquid by No. 23 and No. 26 syringe needles.By in hematimeter, under the situation that has Trypan Blue (0.4% solution, Sigma, catalog number (Cat.No.) T-8154), count, measure cell density and vigor.

In the inoblast perfect medium, with former generation inoblast with 1 * 10 ⁶Viable cell/T75cm ²The cell density of tissue culture flasks is at 38.5 ℃/5% CO ₂Following enlarged culturing.Cultivate after 3 days, perhaps before cell converges, (do not contain Ca with 1 * D-PBS ²⁺And Mg ²⁺) washing flask 1 time, and with 10ml cell dissociation damping fluid at room temperature incubation 5-10 minute, gather in the crops inoblast.With dissociating of inverted microscope range estimation monitoring cell.In this step, carefully guarantee cell mass to be disperseed by grinding.After washing and quantizing, dissociated inoblast can be used for the gene targeting test at any time.These cells also can be frozen preservation for long-term preservation.

HAC is imported in the bovine fetal fibroblast

Adopt chromosome transfer (the Nature Biotech.18:1086-1090 such as Kuroiwa of minicell mediation, 2000), Δ HAC and Δ Δ HAC are transferred to Chinese hamster ovary (CHO) cell from described DT40 cell hybrid, in the F12 (Gibco) that has added 10%FBA (Gibco), 1mg/mlG418 and 0.2mg/ml hygromycin B, in 37 ℃ and 5% CO ₂The following CHO that contains Δ HAC that cultivates clones (" D15 clone ").D15 is cloned in 12 T25 flasks carries out enlarged culturing.When degree of converging reaches 80-90%, in substratum, add Colchiceinamidum (Sigma) with the concentration of 0.1 μ g/ml.After 3 days, substratum is replaced by the DMEM (Gibco) that has added 10 μ g/ml cytochalasin Bs (Sigma).With 8, the centrifugal flask of 000rpm 60 minutes is collected minicell.Allow minicell pass through 8-, 5-and 3-μ m filter (Costar) comes purifying, be resuspended in then in the DMEM substratum.As described below, described minicell and ox inoblast are merged.

In the α-MEM (Gibco) that has added 10%FBS (Gibco), in 37 ℃ and 5%CO ₂Down bovine fetal fibroblast is cultivated.In the T175 flask, inoblast is carried out enlarged culturing.When degree of converging reached 7080%, the trypsinase with 0.05% made cell separate with flask.With DMEM substratum washing inoblast twice, be layered on then on the described minicell suspension.With 1, the centrifugal described minicell of 500rpm-inoblast suspension according to manufacturer's scheme, was added to PEG1500 (Roche) in the cell precipitation after 5 minutes, made minicell and ox inoblast merge.After the fusion, fused cell is loaded on the flat board of 24-hole, in having added the α of 10%FBS-MEM substratum, cultivated 24 hours.Replace this substratum with the substratum that contains 0.7mg/ml G418 then.After about 2 weeks of growth in the presence of the G418 microbiotic, select fused cell with G418 resistance.As described below, these G418 resistance clones are used for nuclear transplantation.

Similarly, by MMCT, Δ Δ HAC is transferred to the bovine fetal fibroblast from CHO clone C13.Selected G418 resistance clone is used to nuclear transplantation.

Nuclear transplantation, activation and embryo culture

Basically implement nuclear transplantation method (Cibelli etc., Science 1998:280:1256-1258) as previous description.After maturation about 18-20 hour (hpm), with the ovocyte stoning of maturation in vitro, (Hoechst 33342, Sigma) mark, checking karyomit(e) removal under ultraviolet ray with the benzyl imines.(San Diego CA), merges these kytoplasms-donorcells doublet for Electrocell manipulator 200, Genetronics by using lasting 20 seconds single electricimpulse of 2.4kV/cm.After 3-4 hour, take out one 25% subgroup at random of all doublets that are transferred, the nucleus that is transferred by two benzyl imines marks confirms to merge.When 30hpm, with ovocyte and contrast method as described earlier (Lin etc., the Mol.Reprod.Dev.1998:49:298-307 of reconstruct; Presicce etc., Mol.Reprod.Dev.1994:38:380-385), (CalBiochem, San Diego CA) and with the ACM substratum that contains 10 μ g cycloheximides and 2.5 μ g cytochalasin D (Sigma) activate 6 hours to activate 4 minutes with Calcium ionophore (5 μ M).After the activation, the washing ovum is 5 times in HEPES buffering Chinese hamster embryo culture medium (HECM-Hepes), place 4 hole tissue culturing plates to cultivate then, contain mouse fetal inoblast and 0.5ml embryo culture medium in this culture plate, and cover the mineral oil (Sigma) of 0.2ml through embryo's check through irradiation.Place 25-50 piece of embryo in each hole, under 38.5 ℃, 5%CO ₂Atmosphere surrounding in cultivate.The 4th day, in substratum, add 10%FCS.

Embryo transfer

At the 7th day and the 8th day, respectively the nuclear transplantation blastaea is transplanted in the receptor cow of synchronization of the 6th day and the 7th day.Adopt disposable injection Lutalyse (Pharmacia ﹠amp; Upjohn, Kalamazoo MI), then checks to oestrus to make the receptor cow synchronization.The existence of gestation is checked by ultrasonic imaging in after embryo transfer the 30th day and the 60th day, after this, checks by rectal palpation in per 30 days, up to 270 days.The reservation situation of HAC in the fetus of these oxen is summarized in the table 2, and described in further detail in following chapters and sections.

Table 2: the HAC in the ox fetus keeps the summary of situation

Contain the importing of the segmental HAC of human chromosome #14

With with the essentially identical method of aforesaid method, SC20 fragment, human chromosome #14 fragment (" hchr.14fg " contains the Ig heavy chain gene) are imported in the fetal fibroblast.Also can adopt any other conventional chromosome transfer method that this HAC or the another kind of HAC that contains people Ig gene are inserted in the donorcells.Resulting donorcells can be used for conventional nuclear transfer technology, and those for example above-mentioned technology have the transgenic ungulates of HAC with production.

By the pregnant situation of 28 pregnancy receptors of ultrasound investigation, transplanted clone embryos for these acceptors from the cell that contains chr.14fg.The result is summarised in the table 3.

If desired, can be used to second from the HAC fetus of gained or HAC offspring's cell and take turns nuclear transplantation, to produce other clone offsprings.Cell from initial HAC fetus or HAC offspring also can be frozen, and forms a kind of clone, originates as the donorcells that other HAC ungulates produce.

Table 3: use the gestation of donorcells in the time of 40 days that contains hchr.l4fg

Clone ID	The acceptor quantity of transplanting	Gestation in the time of 40 days (%)
Clone ID	The acceptor quantity of transplanting	Gestation in the time of 40 days (%)	2-1	8	3(38)
4-2	10	0(0)	2-1	8	3(38)
4-2	10	0(0)	4-1	5	0(0)
4-1	3	1(33)	4-1	5	0(0)
4-1	3	1(33)	2-1	2	1(50)
Amount to	28	5(18)	2-1	2	1(50)

Pregnancy rate is lower than expection.This is because the extremely unusual hot weather during embryo transfer.

As shown in accompanying drawing 27, the pregnancy rate of carrying the embryo of HAC seems suitable with non-transgenic clone's pregnancy rate.Recently, an acceptor of nourishing Δ Δ HAC calf is given birth to the healthy young baby of a work.Other acceptors will be produced in the ensuing several months.

The proof that people's heavy chain gene seat is reset and expressed in Δ HAC ox fetus

When each pregnant fate, take out clone's Δ HAC transgenic cattle fetus, analyze wherein existence, rearrangement and the expression of people's immunoglobulin loci.To analyzing from the spleen of one of these fetuses and the genomic dna and the cDNA of non-Lymphoid tissue (liver and brain) acquisition, show existence, rearrangement and the expression of described Δ HAC by RT-PCR.

In Δ HAC fetus, there are people's heavy chain and/or light chain

In order to determine in Δ HAC fetus, whether to exist people's heavy chain and light chain, from Δ HAC fetus, separate liver dna, whether there is the genomic dna of coding people's heavy chain and light chain by pcr analysis.

In order to detect genome heavy chain DNA, use following primer: VH3-F5 '-AGT GAGATA AGC AGT GGA TG-3 ' (SEQ ID NO:1) and VH3-R5 '-CTT GTGCTA CTC CCA TCA CT-3 ' (SEQ ID NO:2).The primer that is used to detect lambda light chain DNA is: IgL-F5 '-GGA GAC CAC CAA ACC CTC CAA A-3 ' (SEQ IDNO:3) and IgL-R5 '-GAG AGT TGC AGA AGG GGT YGA CT-3 ' (SEQID NO:4).The PCR reaction mixture contains 18.9 μ l water, 3 μ l10 * Ex Taq damping fluid, 4.8 μ ldNTP mixtures, 10pmol forward primer and 10pmol reverse primer, 1 μ l genomic dna and 0.3 μ l Ex Taq.The incubation reaction mixture carries out 38 PCR circulation under the following conditions: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ of 10 second, 56 ℃ of 30 second and 72 ℃ of 30 second.

As shown in Figure 5, fetus #5968,6032 and 6045 all contain people's heavy chain (μ) and light chain (λ) locus.Fetus #5996 only contains people's heavy chain gene seat.Fetus #5983 does not contain people's heavy chain gene seat, and may not contain people's light chain gene seat.Fetus #5846 does not contain any human sequence.Therefore, fetus #5983 and 5846 may not keep HAC.These results show that Δ HAC can stably remain into the 58th day of gestation in ox.

In Δ HAC fetus #5996, there is people C μ exon

The primer that mRNA transcribes that is specific to that comprises C μ 3 and C μ 4 parts is used to detect the transcript that whether Δ HAC exists and whether express coding people μ locus constant region among the fetus #5996.

RT-PCR for the genome constant region of people μ heavy chain analyzes, use primer " CH3-F1 " (5 '-acc acc tat gacagc gtg ac-3 ', SEQ ID NO:5) and " CH4-R2 " (5 '-gtggca gca agt aga cat cg-3 ', SEQ ID NO:6), generate the RT-PCR product of 350bp.This pcr amplification by carrying out 95 ℃ of following sex change insulations at first in 5 minutes.Then, 95 ℃ following 1 minute, following 2 minutes of 59 ℃ of following 1 minute and 72 ℃ carry out 35 round-robin sex change, annealing and amplifications.Then, in 72 ℃ of following insulation reaction mixtures 10 minutes.As described below, detect the Niu Chonglian (accompanying drawing 7) of rearrangement with primer I 7L and P9.As internal contrast, with primer " GAPDH forward " (5 '-gtcatcatc tct gcc cct tct g-3 ', SEQID NO:7) and " GAPDH is reverse " (5 '-aac aac ttc ttg atg tca tca t-3 ', SEQID NO:8) detect the level of GAPDH RNA.For the amplification of GAPDH RNA,, then, carry out 35 round-robin insulations in 95 ℃ 1 minute, 55 ℃ 1 minute and 72 ℃ 2 minutes at 95 ℃ of following insulation samples 5 minutes.Then, 72 ℃ of following incubation mixtures 7 minutes.

This analysis revealed is analyzed the RT-PCR of fetus #5996 spleen and to be produced a band (swimming lane 3), its with contrast people spleen cDNA (swimming lane 4) with from the amplified production of cDNA (swimming lane 5) generation of Δ HAC gomphosis mouse be complementary (accompanying drawing 6).In non-Lymphoid tissue: do not detect this band in cattle liver (swimming lane 1) or the ox brain (swimming lane 2).The house-keeping gene GADPH that successfully increased in liver (swimming lane 10 of accompanying drawing 6) and brain (swimming lane 6 of accompanying drawing 7) has shown the ability of these tissue support RT-PCR.

The rearrangement of gestation ox heavy chain gene seat in the time of 77 days

Detect Δ HAC fetus #5996, detect it and whether experienced the immunoglobulin heavy chain gene seat and reset required recombination system and express and activate necessary growth course.Analyze for this, carry out conventional RT-PCR and analyze to detect whether have the mRNA transcript that coding μ-VH resets.Use primer " 17L " (5 '-ccc tcc tct ttg tgc tgt ca-3 ', SEQID NO:9) and " P9 " (5 '-cac cgt get etc ate gga tg-3 ', SEQ ID NO:10), by the RNA of RT-PCR analytical separation from the #5996 of fetus spleen, liver and brain.At 95 ℃ of following insulation PCR reaction mixtures 3 minutes, adopt following condition then: 95 ℃ 1 minute, 58 ℃ of 1 minute and 72 ℃ 2 minutes carry out 35 round-robin sex change, annealing and amplifications.Then, in 72 ℃ of following insulation reaction mixtures 10 minutes.

The swimming lane 5 of accompanying drawing 7 shows, obtains expecting the rearrangement Niu Chonglian amplified production (450 of size

bp)。This product is moved to and is contrasted on the suitable position, the position of ox C μ heavy chain cDNA amplified production (swimming lane 7), and known described contrast contains the sequence corresponding to the Niu Chonglian transcript of resetting.As expected, the heavy chain of rearrangement is expressed (swimming lane 5) in spleen, but nothing expression in the brain (swimming lane 2) of this etap and liver (swimming lane 3).

Rearrangement and the expression of people's heavy chain gene seat in Δ HAC fetus #5996

Comprise the DNA section of C μ and VH area part, the rearrangement and the expression of reference's heavy chain gene seat by amplification.Determine with the primer that is specific to the rna transcription thing that comprises C μ (C μ 1) and VH (VH3-30) part whether exist and contain the RNA heavy chain thing (accompanying drawing 8) of resetting people C μ-VDJ sequence.

Analyze for this RT-PCR, primer " C μ 1 " (5 '-cag gtg cag ctg gtg gag tctgg-3 ', SEQ ID NO:11) and ＂ VH3-30 ＂ (5 '-cag gag aaa gtg atg gagtc-3 ', SEQ ID NO:12) be used to prepare the RT-PCR product of 450bp.This RT-PCR is performed as follows: reaction mixture in 95 ℃ of down insulations 3 minutes, was then carried out 95 ℃ of 40 round-robin 30 minutes, and 69 ℃ of 30 minutes and 72 ℃ 45 minutes and 72 ℃ of insulations of following 10 minutes circulate.This RT-PCR product is increased with the same primers as case again, 95 ℃ following 3 minutes one insulation circulation, 95 ℃ 1 minute, 59 ℃ of 1 minute and 72 ℃ of 40 insulation circulations of 1 minute, and 72 ℃ of insulations of following 10 minutes circulate.As internal contrast, as mentioned above, carry out the RT-PCR amplification of GAPDH.

Gel in the accompanying drawing 8 shows, the RT-PCR from the spleen of fetus #5996 analyzed produce a band (swimming lane 5), and the amplified production that produces with personnel selection spleen cDNA (swimming lane 4) or Δ HAC gomphosis mouse spleen cDNA (swimming lane 1) is complementary.In non-Lymphoid tissue: do not detect this band in cattle liver (swimming lane 2) or the ox brain (swimming lane 3).As positive control, the amplification of GADPH RNA (swimming lane 8 and 9) shows the ability of these tissue support RT-PCR.

Also use primer CH3-F3 (5 '-GGA GAC CAC CAA ACC CTC CAAA-3; SEQ ID NO:13) and CH4-R2 (5 '-GTG GCA GCA AGT AGACATCG-3 '; SEQ ID NO:14), analyzes, confirmed rearrangement and the expression of people's heavy chain district in fetus #5996 by RT-PCR.These PCR reaction mixtures contain 18.9 μ l water, 3 μ l10 * Ex Taq damping fluid, 4.8 μ l dNTP mixtures, 10pmol forward primer and 10pmol reverse primer, 1 μ lc DNA and 0.3 μ l Ex Taq.The incubation reaction mixture carries out 40 PCR circulation under the following conditions: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ of 10 second, 60 ℃ of 30 second and 72 ℃ of 30 second.

Shown in the

swimming lane

6 and 7 of accompanying drawing 9, identical with the segmental size of montage constant region from two positive controls from the extension increasing sequence of the spleen of fetus #5996, positive control is: from the sample (swimming lane 8) and the Δ HAC gomphosis mouse spleen cDNA (swimming lane 9) of people's spleen.As expected, the negative control from normal mouse spleen and cattle spleen does not contain extension increasing sequence (

swimming lane

1 and 2).Do not contain and pass through the montage sequence of the people μ CH fragment amplification of a size of montage from the liver of fetus #5996 and the sample of brain, but contain the extension increasing sequence (

swimming lane

3,4 and 5) of the not montage genomic fragment that derives from the genomic dna that pollutes the RNA sample.

The VDJ of people's heavy chain gene seat in Δ HAC fetus resets

Also carried out the RT-PCR analysis, in Δ HAC fetus #5996, the VDJ rearrangement has taken place with further confirmation heavy chain gene seat.Carry out nested RT-PCR, first reaction use primer C μ-1 (5 '-CAGGAGAAAGTGATGGAGTC-3 '; SEQ ID NO:15), second reaction use primer C μ-2 (5 '-AGG CAG CCA ACG GCC ACGCT-3 '; SEQ ID NO:16), all use in two reactions primer VH3-30.3 (5 '-CAG GTG CAGCTG GTG GAG TCTGG-3 '; SEQ ID NO:17).The RT-PCR reaction mixture contains 18.9 μ l water, 3 μ l10 * Ex Taq damping fluid, 4.8 μ ldNTP mixtures, 10pmol forward primer and 10pmol reverse primer, 1 μ l cDNA and 0.3 μ l Ex Taq.Carry out RT-PCR, in first reaction, carry out 38 circulations under the following conditions: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ of 10 second, 65 ℃ of 30 second and 72 ℃ of 30 second.For second reaction, use primer VH3-30.3 and C μ-2 (5 '-AGG CAG CCA ACG GCCACGCT-3 '; SEQ ID NO:16), carry out 38 circulations under the following conditions: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ of 10 second, 65 ℃ of 30 second and 72 ℃ of 30 second.

Shown in the

swimming lane

6 and 7 of accompanying drawing 10, the spleen of fetus #5996 is carried out RT-PCR analyze, produce size and the same heavy chain bands of positive control in swimming lane 8 and 9.The rearrangement DNA (swimming lane 3 and 5) that contains some contaminative from the sample of the liver of fetus #5996 and brain.Negative control in the

swimming lane

1 and 2 produces big or small incorrect band.

Confirm that by order-checking Δ HAC resets

Obtain cDNA from the RNA reverse transcription of the spleen of Δ HAC fetus #5996,, and on sepharose, carry out electrophoresis with this cDNA of primer amplification of the people μ heavy chain that is specific to rearrangement.Cut the band that amplification is obtained by C μ l-VH3-30 primer from gel.From the cDNA of this band recovery amplification and with its clone.Purifying detects the DNA that clones for the people μ male of resetting from gained through PCR, and to its check order (accompanying drawing 11A).

Surpass 95% from the sequence of this Δ HAC fetus and the homology of the known person sequence of heavy chain more than 20 kinds.For example, a district of the μ chain of the anti-streptococcus pneumoniae antibody of people and this sequence has 97% homology (accompanying drawing 11B).

Analyze by the spleen of fetus #5996 being carried out RT-PCR, then increase again, must arrive other sequences from people's heavy chain of rearrangement with this primer with primer C μ-2 and VH3-30.3.CHROMA SPIN post (CLONETECH) purifying RT-PCR product, and, it is cloned into (Invitrogen) on the pCR2.1TA cloning vector according to manufacturer's scheme.Carry out dyestuff terminator sequence reaction (ABI AppliedSystem) in the reaction mixture of 10 μ l volumes, this mixture is made up of BigDye terminator reaction mixture (3 μ l), template plasmid (200ng) and C μ-2 primer (1.6pmol).Adopt the ABI3700 sequenator to check order.For this analysis, carry out 25 circulations under the following conditions: 96 ℃ 1 minute, 96 ℃ 10 second, 55 ℃ of 5 second and 60 ℃ 4 minutes.

Identify people's heavy chain transcript of at least two kinds of rearrangements, it is VH3-11/D7-27/JH31Cp and VH3-33/D6-19/JH21Cp (accompanying drawing 12A and 12B).These results prove that in the spleen of fetus #5996, the VDJ that people μ heavy chain gene seat takes place among the described Δ HAC resets.From same fetus, identify the sequence of heavy chain of more than a kind of rearrangement, proved that also Δ HAC fetus can produce the various human immunoglobulin sequences.

Rearrangement and the expression in Δ Δ HAC fetus of people's heavy chain and light chain gene seat

At the pregnant fate of difference, from receptor cow, take out the clone fetus that comes from bovine fetal fibroblast Δ Δ HAC chromosome transfer.Analyze existence and the rearrangement of the HAC of these fetus carrier heavy chain immunoglobulins and lambda light chain gene seat.To from studies show that of the locus DNA of these tissues, in some fetus, there are human immunoglobulin heavy chain and light chain.The cDNA of the spleen that derives from these fetuses checked show that heavy chain immunoglobulin and light chain gene seat exist and reset and express in some fetus.Facs analysis also proved, people's lambda light chain albumen is expressed on the spleen lymphocyte surfaces of two fetuses therein.

In Δ Δ HAC fetus, there are people's heavy chain and light chain gene seat

In order to determine whether Δ Δ HAC fetus has kept people's heavy chain and light chain gene seat, the genomic dna from the liver of the fetus #5442A of 58 days fetus #5580,57 days fetus #5848 and 91 days and 5442B has been carried out pcr analysis.The PCR primer that is used to detect the heavy chain gene seat be VH3-F (5 '-AGT GAG ATA AGC AGT GGATG-3 '; SEQID NO:18) and VH3-R (5 '-CTT GTG CTA CTC CCA TCACT-3 '; SEQID NO:19), the primer that is used to detect light chain be IgL-F (5 '-GGA GAC CAC CAAACC CTC CAAA-3 '; SEQ ID NO:20) and IgL-R (5 '-GAG AGT TGCAGA AGG GGT YGACT-3 '; SEQ ID NO:21).The PCR reaction mixture contains 18.9 μ l water, 3 μ l, 10 * Ex Taq damping fluid, 4.8 μ l dNTP mixtures, 10pmol forward primer and 10pmol reverse primer, 1 μ l genomic dna and 0.3 μ l Ex Taq.Followingly carry out 38 PCR circulation: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ of 10 second, 56 ℃ of 30 second and 72 ℃ of 30 second (attached Figure 13 and 14).

Shown in attached Figure 13 and 14,58 days fetus #5580 of positive control contains people's heavy chain and two locus of light chain immunoglobulin (Ig).In addition, 91 days fetus #5442A and 5442B also contain heavy chain and two locus of light chain (accompanying drawing 14).On the contrary, fetus #5848 does not contain any people's gene seat, may not contain Δ Δ HAC yet.These results show that Δ Δ HAC can stably remain into the 91st day of gestation in ox.

Rearrangement and the expression of people's heavy chain gene seat in Δ Δ HAC fetus #5442A

Detect the expression of people's heavy chain rna transcription thing in Δ Δ HAC fetus #5542A of rearrangement with RT-PCR.Used RT-PCR primer be CH3-F3 (5 '-GGA GAC CACCAA ACC CTC CAAA-3 '; SEQID NO:22) and CH4-R2 (5 '-GAG AGTTGC AGA AGG GGT GACT-3 '; SEQ ID NO:23).The RT-PCR reaction mixture contains 18.9 μ l water, 3 μ l, 10 * Ex Taq damping fluid, 4.8 μ l dNTP mixtures, 10pmol forward primer and 10pmol reverse primer, 1 μ l cDNA and 0.3 μ l Ex Taq.Followingly carry out 40 RT-PCR circulation: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ of 10 second, 60 ℃ of 30 second and 72 ℃ of 30 second.

The

swimming lane

4 and 5 of accompanying drawing 15 contains the amplification montage μ CH sequence from the spleen of fetus #5442A, and its size is big or small similar to described positive control.These results show that fetus #5442A expresses a kind of μ heavy chain transcript of rearrangement in its spleen.Also observe fuzzy band in the zone of the locus sequence of not montage, they are that the genomic dna amplification of the pollution from dna sample obtains.Do not produce the amplification retracing sequence from the check sample of the liver of fetus #5442A and brain and expect big or small band.

Rearrangement and the expression of people's heavy chain gene seat in Δ Δ HAC fetus #5868A

Detect the expression of people's heavy chain rna transcription thing in the spleen of 119 days Δ Δ HAC fetus (fetus #5868A) of a gestation of rearrangement with RT-PCR.The primer that is used for this analysis be VH30-3 (5 '-cag gtg cag ctg gtg gag tctgg-3 '; SEQ ID NO:24) and CM-1 (5 '-cag gag aaa gtg atg gagtc-3 '; SEQ ID NO:25).In addition, primer " on the GAPDH " (5 '-gtc atc atc tct gcc cct tctg-3 '; SEQ ID NO:26) and " under the GAPDH " (5 '-aac aacttc ttg atg tea tca t-3 '; SEQ ID NO:27) the GAPDH control transcripts that is used to increase.For this pcr analysis, reaction mixture 95 ℃ of down insulations 5 minutes, by 95 ℃ of 1 minute, 58 ℃ of insulations insulation 1 minute and 72 ℃ insulations 2 minutes down down down, is carried out the circulation of a plurality of sex change, annealing and amplification then.Then, mixture is incubated 10 minutes down at 72 ℃.

The swimming lane 3 of accompanying drawing 16 contains analyzes the RT-PCR product that is produced to Δ Δ HAC fetus #5868A.This RT-PCR product has the expection size (470bp) of people's heavy chain of amplification rearrangement, and moves in gel and the known identical position of containing corresponding to the sequence of people's transcript of resetting of contrast cDNA.In contrast, when increasing with primer, the spleen cDNA of Δ Δ HAC fetus #5868A and normal ox cDNA sample all produce a kind of product, have proved that this cDNA supports the ability (swimming lane 7 and 8) of amplification.

Rearrangement and the expression of people λ locus in Δ Δ HAC fetus #5442A and 5442B

Use is specific to the primer of the transcript that comprises people λ part, detects the rna transcription thing from people's lambda light chain gene seat of resetting.

Analyze for the RT-PCR shown in the accompanying drawing 17, use primer C λ 1 (5 '-GGG AATTCG GGT AGA AGT TCA CTG ATCAG-3 '; SEQ ID NO:28), C λ 2-3 (5 '-GGG AAT TCG GGT AGA AGT CAC TTA TGA G-3 '; SEQ IDNO:29) and C λ 7 (5 '-GGG AAT TCG GGT AGA AGT CAC TTA CGAG-3 '; Molar mixture such as grade SEQ ID NO:30) and primer V λ 1 LEA1 (5 '-CCCCCA AGC TTR CCK GST YYC CTC TCC TC-3 '; SEQ ID NO:31).Described RT-PCR reaction mixture contains 18.9 μ l water, 3 μ l, 10 * Ex Taq damping fluid, 4.8 μ l dNTP mixtures, 10pmol forward primer and 10pmol reverse primer, 1 μ l cDNA and 0.3 μ l Ex Taq.The condition of RT-PCR is as follows: 85 ℃ 3 minutes, 94 ℃ 1 minute, in 98 ℃ of 10 second, 60 ℃ of 30 second and 72 ℃ 1 minute carry out 40 circulations.

As shown in Figure 18, this RT-PCR analyze also use primer V λ 3LEA1 (5 '-CCCCCA AGC TTG CCT GGA CCC CTC TCT GG-3 '; SEQ ID NO:32), V λ 3JLED (5 '-ATC GGC AAA GCT TGG ACC CCT CTC TGG CTCAC-3 '; SEQ ID NO:33) and V λ BACK4 (5 '-CCC CCA AGC TTC TCGGCG TCC TTG CTT AC-3 '; Molar mixture such as grade SEQ ID NO:34) and primer C λ 1 (5 '-GGG AAT TCG GGT AGA AGT TCA CTG ATCAG-3 '; SEQID NO:35), C λ 2-3 (5 '-GGG AAT TCG GGT AGA AGT CAC TTA TGAG-3 '; SEQ ID NO:36) and C λ 7 (5 '-GGG AAT TCG GGT AGA AGT CACTTA CGA G-3 '; SEQ ID NO:37) molar mixture such as grade.The reaction conditions of described RT-PCR is with above identical about accompanying drawing 7 described conditions.

The swimming lane 6 of accompanying drawing 17 and 7 and the

swimming lane

4 and 5 of accompanying drawing 18 contain RT-PCR product from the spleen of fetus #5442A, its size is similar with the positive control band, this shows and has the light chain rna transcription thing of resetting in this fetus.Spleen sample from fetus #5442B produces very faint sizeable band (invisible on this photo).This RT-PCR product shows that fetus #5442B also expresses the light chain immunoglobulin (Ig) transcript of resetting in its spleen.As desired, from the sample of #5442A and the 5442B brain rearrangement lambda light chain transcript of expressing human not.

Rearrangement and the expression of people λ locus in Δ Δ HAC fetus #5868A

In Δ Δ HAC fetus #5868A, also detect the rna transcription thing of people's lambda light chain gene seat of rearrangement.For this analysis, use the primer that is specific to the transcript amplification that comprises people λ part, the expression of the Δ Δ HAC coding of the transcript of the people λ locus part that detecting encodes resets.Use primer VL1 LEAI during this is analyzed (5 '-ccc ccaagc ttR ccK gSt Yyc ctctcctc-3 '; SEQ ID NO:38), and primer CL1 (5 '-ggg aat tog ggt aga agtcac tga tcag-3 '; SEQ ID NO:39), CL2-3 (5 '-ggg aat tog ggt aga agt cactta tgag-3 '; SEQ ID NO:40) and CL7 (5 '-ggg aat tog ggt aga agt cac ttacgag-3; SEQ ID NO:41) molar mixture such as grade.For this RT-PCR reaction, reaction mixture is incubated 5 minutes down at 95 ℃, be incubated 1 minute down at 95 ℃ then, be incubated the circulation of carrying out a plurality of sex change, annealing and amplification in 2 minutes down 60 ℃ of insulations 1 minute with at 72 ℃.Then, mixture is incubated 10 minutes down at 72 ℃.

This analytical proof is from spleen cDNA (swimming lane 4 of the accompanying drawing 19) generation of Δ Δ HAC#5868A and the RT-PCR product of TC mouse spleen cDNA (swimming lane 6) the identical size of positive control.Use derives from brain or the liver cDNA (being respectively swimming lane 2 and 3) of Δ Δ HAC#5868A, does not detect this RT-PCR product.With primer " on the GAPDH " and " under the GAPDH " the house-keeping gene GAPDH (swimming lane 8 and 10) that successfully increases, show that in these tissues each can both support RT-PCR.

Confirm that by order-checking Δ Δ HAC resets

Use primer C λ 1, C λ 2-3 and C λ 7 etc. molar mixture and primer V λ lLEAl, perhaps use primer V λ 3LEAl, V λ 3JLEAD and V λ BACK4 etc. molar mixture and primer C λ 1, C λ 2-3 and C λ 7 etc. molar mixture, the spleen sample of fetus #5442A is carried out RT-PCR analyzes.With CHROMA SPIN post (Clontech) purified pcr product, and, it is cloned in the pCR2.1TA cloning vector (Invitrogen) according to manufacturer's scheme.Use C λ 1, C λ 2-3 and C λ 7 etc. molar mixture, carry out dyestuff terminator serial response (ABI Applied System).Carried out 25 circulations in 4 minutes with 96 ℃ 1 minute, 96 ℃ 10 second, 55 ℃ of 5 second and 60 ℃.Contain BigDye terminator reaction mixture (3 μ l), template plasmid (200ng) and C λ 1, C λ 2-3 and C λ 7 primers (1.6pmol) in the 10 μ l reaction mixtures.With ABI 3700 sequenator analyze reaction mixtures.

Identify people's lambda light chain transcript (V1-17/JL3/Ck and V2-13/JL2/Ck) of at least two kinds of rearrangements.These results prove that VJ takes place people's lambda light chain gene resets (accompanying drawing 20 and 21) in the Δ Δ HAC of the spleen of fetus #5442A.

The facs analysis of people's lambda light chain and the Niu Chonglian expression in Δ Δ HAC fetus #5442A and 5442B

Analysis is from the expression of people's lambda light chain and ox heavy chain protein in the spleen lymphocyte of Δ Δ HAC fetus #5442A and 5442B.The anti-people λ antibody (accompanying drawing 22C and 22D) of these cells and phycoerythrin mark, the anti-ox IgM antibody (accompanying drawing 22D and 22H) of FITC mark or do not have antibody (accompanying drawing 22A, 22B, 22E and 22F) 4 ℃ of down reactions 20 minutes.Use then and added the PBS washed cell twice of 2%FCS, and on the FASCalibur cell sorter, analyze.Contrast electronic settings gate (electronically set thegates) with no antibody, the percentage of the cell of calculating and described antibody response.These percentage are presented at the below of each frequency curve.Fetus #5442A (accompanying drawing 22A-22D) and fetus #5442B (accompanying drawing 22E-22H) expressing human lambda light chain albumen and ox heavy chain protein.

The expression of people's antibody protein in the HAC calf

As mentioned above, a kind of novel method is developed the calf (accompanying drawing 37) of production chromosome transfer (Tc).This method overcome since former generation the ox inoblast only have an appointment 35 population doublings finite life and must have a large amount of DNA insets to be imported into and to maintain restriction in the donorcells.Especially, with human artificial chromosome (HAC) carrier complete people Ig heavy chain (IgH) that is not rearranged and lambda light chain (IgR) locus are imported to the Niu Yuandai inoblast.Fetus by the preparation clone restores and the selected fibroblast cloning that increases.Select clone's fetal cell also to clone to produce the Tc calf of four health, it is functionally reset heavy chain and light chain people Ig locus and produces people's polyclonal antibody again.These results prove, use the HAC carrier to produce the feasibility of breeding transgenic livestock.More importantly be, the Tc ox that contains people Ig gene can be used to produce new people's polyclone therapeutical agent, can be applicable to infect the scope of resisting bio-terrorism from the prevention antibiotics resistance.These methods are described hereinafter in further detail.

Adopt MMCT technology described here, HAC is imported to the bovine fetal fibroblast from the CHO clone.Inoblast is placed with under the screening of G418 (700pg/ml) to the neo genetic marker on the HAC carrier, up to the clone occurring.In this step, avoid antibiotic-screening completely and based on the screening of DNA, so that the cytodifferentiation before the nuclear transplantation minimizes.Come selected clone according to growth and morphology, and carry out nuclear transplantation (Lucier etc., J.Immunol.161:5438-5444,1998) as previous description.Because this cell is useful to nuclear transplantation in several day only, by produce clone fetus restore and increase after carry out last screening.

The proportional range of growing the blastaea stage is between 17-21%, and 40 days pregnancy rate does not have difference between the clone between 22-50%.At 56-58 days, take out four Δ HAC fetuses and two Δ Δ HAC fetuses, rebuild fibroblast, freezing preservation is used for further analyzing and nuclear transplantation.By the genome PCR (accompanying drawing 38B) of G418 resistance (accompanying drawing 38A) and IgH and Ig λ locus, estimate HAC at the fetus of collecting and 7 conservation rates in the fibroblast of the fetus of collection in 77-119 days from 56-58 days.9 in 13 fetuses have resistance to G418, and 8 demonstrations in them have people IgH and Ig λ locus.Three Δ HAC (#5968, #6032 and #6045) and 56-58 days fetus of a Δ Δ HAC (#5580) male are used for cloning and producing the offspring.Analyze by RT-PCR, then amplified production is checked order, come expression and the rearrangement of appraiser Ig locus in 5 male 77-119 days fetus.Expressing human IgH and Ig λ gene (accompanying drawing 38C) and shown that correct V (D) J resets the evidence of (accompanying drawing 38D) in all fetuses.

By 37 acceptors, Ke Long Δ HAC clone has been produced 1 male (clone #6045) and 5 female calves (clone #5968 and #6032) (16%, accompanying drawing 39A) again.Two derives from calf death in postnatal 48 hours of clone #6032.A calf is from deregenerative Δ Δ HAC cell.5 whole calves are all very healthy, and phenotype is normal.Analyze (accompanying drawing 39B and 39C) by G418 selection, genome PCR and fluorescence in situ hybridization (FISH), determine in all calves, to have kept HAC.Fish analysis is the result show, HAC is retained as independent karyomit(e), and the cell proportion of reservation HAC is 78-100%.Between peripheral blood lymphocyte (PBLs, 91%) and inoblast (87%), do not observe the difference of tangible retention rate.Donorcells is that may to have than donorcells be the higher retention rate of #5968 (being respectively 97% and 86%) to #6045.Ironically, the retention rate in the Tc ox may be observed higher in mouse than the previous inventor.In order to determine whether people Ig locus is rearranged and expresses, and the inventor has carried out the RT-PCR analysis to PBLs in calf and fetus.The inventor observes people IgH and Ig λ expression of gene in PBLs, and determines the diversity (table 4) of all components of people IgH and Ig λ by sequencing analysis.The representational sequence of one cover has shown the V that is distributed on the locus _H/ V λ, D _HAnd J _HThe extensive utilization of/J λ section.In Ig λ transcript, observed from V _H1 and V _HThe frequent utilization of 3 V section, this utilization and V _HSection utilizing in the people is similar.In IgH and Ig λ transcript, also observe the deletion of interpolation that non-kind is a Nucleotide (N-interpolation) and Nucleotide.This produces highly variation in the 3rd complementary determining region (CDR3s) of heavy chain and light chain.In addition, determine, in 5 of 7 calves, detect the people Ig albumen (in newborn calf, Ig expresses very low usually, to such an extent as to can not detect) of 13-258ng/ml in the blood sample of before the colostrum of feeding, gathering by solid phase ELISA.These data show, adopt clone's strategy again, can carry out HAC effectively and shift in primary cell, and entrained people Ig gene can correctly be processed among the HAC, and expressed on Tc calf camber diversity ground.

Result of the present invention shows that karyomit(e) clone, chromosome transfer and the somatocyte combination of clone technology again can be used to produce the healthy calf that has kept the genetically modified HAC carrier of the genome that carries the Mb size.In addition, these technology are used to prove the transfer and the reservation of the complete genome seat of people IgH and Ig λ gene in ox.Ironically, verified in the kind that has with people or the diverse immunophysiology of mouse, these two human immunoglobulin genes that locus carries out correct processing and expressive function rearrangement.It is useful that this HAC system gathers for the human protein (for example oxyphorase) of expressing multiple complexity and the big protein that is used for pharmaceutical application.For example, the Tc calf that produces in this research has kept people IgH and Ig λ locus, can be used for the production of people's polyclonal antibody.At present, human polyclonal antibody only can obtain from human blood or blood plasma donor.Therefore, there are the supply of human polyclone product and the restriction of application.The Tc milk cow can be produced a large amount of new polyclone therapeutical agents that are used for the treatment of multiple human diseases by hyperimmunizationization.

Table 4: the repertoire analysis of human immunoglobulin heavy chain in clone's the Tc calf and the light key transcript of λ

By the specific mrna of RT-PCR amplification people μ and λ, clone and check order.Provide 10 independently nucleotide sequences of being connected of V (D) J of each among μ and the λ clone, be divided into V _H/ V λ, D _H, J _H/ J λ and N section, by with disclosed kind be that the homology of sequence is identified (Ig-BLAST).

Embodiment 3: the transgenic ungulates that has the production heteroantibody of sudden change in one or more endogenous antibody

At as described in example 2 above expression heteroantibody and in prion gene, have in the ungulate of sudden change, can reduce the expression of endogenous antibody randomly by the one or more endogenous antibody genes of sudden change.By quantity with respect to functional endogenous heavy chain and light chain gene, increase the quantity of functional heteroimmunity sphaeroprotein heavy chain or light chain gene, the quantity of expressing the B cell of the heteroantibody of expecting (for example human therapeutic antibody) will increase.

In order to produce these transgenic ungulates, can allow the transgenic ungulates mating that contains sudden change in one or more allelotrope of Δ HAC or Δ Δ HAC transgenic ungulates and endogenous immunoglobulin chain (for example μ heavy chain or λ or K light chain).If desired, can allow resulting transgenic ungulates and the transgenic ungulates that (i) in one or two allelotrope of endogenous α-(1,3)-galactosyltransferase, Protein virus and/or J chain nucleic acid, has the transgenic ungulates of sudden change or (ii) contain exogenous J chain nucleic acid (for example people's J chain nucleic acid) carry out mating.Alternately, come the cell (for example fetal fibroblast) of genetic modification by one or more endogenous immunoglobulin genes that suddenly change from Δ HAC or Δ Δ HAC transgenosis fetus.In the possible method of another kind, Δ HAC or Δ Δ HAC are directed to (for example fetal fibroblast) in the cell, and in this cell, endogenous immunoglobulin (μ heavy chain and/or lambda light chain) is by the narrowed deactivation or the deactivation of isozygotying.In arbitrary aforesaid method, can also be by (i) at endogenous α-(1,3)-and import sudden change in one or two allelotrope of galactosyltransferase, Protein virus and/or J chain nucleic acid, preferably knocking out sudden change or (ii) importing external source J chain nucleic acid and coming the genetic mutation cell.Then, the transgenic cell that obtains is used for the nuclear transplantation method, comes the transgenic ungulates of hoping production phase.Exemplary method is below described.

DNA construct

Can use above-mentioned μ heavy chain (accompanying drawing 2A), lambda light chain, κ light chain, α-(1,3)-galactosyltransferase, Protein virus and/or J chain to knock out construct.Alternately, can use following tetracycline resistance μ heavy chain construct (accompanying drawing 3F).Design 4 main coding exons that this construct removes ox μ heavy chain gene seat, but keep complete membrane spaning domain, μ heavy chain gene seat is inactivated.

Assemble the tetracycline construct as follows.The XhoI fragment that will contain the 4.4Kb in the zone that is close to the coding exons 1 is inserted on the XhoI site of pBluescript II SK+.Plasmid pPGKPuro contains puromycin resistance gene, derives from Peter doctor W.Laird, Whitehead Institute, USA.The 1.7KbXhoI fragment subclone that will contain puromycin resistance gene is close to the 4.4Kb fragment and is positioned at its downstream to the SalI site that is present in the polylinker zone.This 1.7Kb tetracycline mark has replaced coding exon CH1, CH2, CH3 and the CH4 of B-IgG heavy chain gene seat.The XbaI fragment in 4.6Kb district that contains the μ locus in these four exon downstreams that are arranged in wild type gene group sequence is added to this construct, as second homologous region.

In order to produce final target practice construct,, generate the subclone of this construct by cut this three kinds of fragments that assemble with NotI and MluI.The restrictive diges-tion of MluI is punctured into 1.4Kb with the 4.6Kb fragment.The NotI site is arranged in polylinker, and can not cut into subclone DNA itself.The MluI site is flat with Klenow fragment benefit, produce flush end.Utilization is present in NotI and the SmaI site in the pBluescript carrier, and NotI/ is mended flat MluI fragment subclone in new Bluescript IISK+ carrier.For gene targeting, with the last carrier of NotI linearizing.

Carry out gene targeting and the inoblast of transfection is carried out drug screening by electroporation

For electroporation, with 1 * 10 ⁷The single-cell suspension liquid of the individual bovine fetal fibroblast that has experienced limited number of times population doublings (for example pressing the described inoblasts that obtain from Δ HAC or Δ Δ HAC transgenosis fetus of embodiment 2) centrifugal 5 minutes with 1200rpm is resuspended in serum-free α-MEM substratum of 0.8ml.With resuspended cell transfer (Invitrogen, cat﹠amp in the 0.4cm electroporation sample pool; Num; P460-50).Then, add the linearizing gene targeting carrier DNA of 30 μ g Restriction Enzymes, and with the 1ml transfer pipet with the contents mixed in the sample pool, carry out an incubation step that room temperature is following 2 minutes then.Sample pool is inserted in the vibration chamber (shaking chamber) of GenePulserII electric perforating system (Biorad), carries out electroporation with 1000 volts and 50 μ F then.Rapidly sample pool is transferred in the tissue culture cabinet, joined in the inoblast perfect medium of about 30ml with the cell of suction pipe with electroporation.Cell is distributed in the tissue culture ware (Corning, cat # 431079) of 30 100mm, rotation makes the cell uniform distribution, then at 38.5 ℃/5%CO lightly ₂Following incubation 16-24 hour.The sucking-off substratum is replaced by the complete inoblast substratum that contains selected selection medicine.Per replacing of carrying out substratum in two days continues 7-14 days altogether.In the chosen process of described medicine, the representational flat board of range estimation monitoring detects the death of cell and the formation of colony.Setting negative control flat board, this negative control flat board contains the inoblast of electroporation when having or not gene targeting carrier, should not produce colony in the chosen process of described medicine.

Select the inoblast colony of drug resistance and carry out cell amplification

After the drug screening step is finished (7-14 days usually), the drug resistance colony is macroscopical visible, at any time can be for transferring in the 48 hole tissue culturing plates to increase.For the secondary transfer process, irising out single colony in the bottom of tissue culture plate with colored marking pen (Sharpie).With 1 * D-PBS (no Ca2 ⁺And Mg ²⁺) washing contains clone's tissue culture plate 2 times, adds the 1:5 diluent of 5ml cell lysis buffer solution then in each flat board.After 3-5 minute room temperature incubation step, single colony begins to separate with the bottom of tissue culture ware.Before colony separates, stop that with P200 pipettor and aerosol suction nozzle (aerosol barrierpipette tip) (200 or 250 μ l) transfers to them respectively in the single hole of 48 hole tissue culture plate.After transfer, with suction pipe pressure-vaccum repeatedly, colony is dissociated fully, add 1ml inoblast perfect medium then.Have drug resistance in order to ensure described cell, continue to carry out drug screening in the stage in whole described 48 holes.At 38.5 ℃/5% CO ₂The following colony that cultivate to shift, and with inverted microscope range estimation monitoring.After 2-7 days, will be near the hole of converging with 1 * D-PBS (no Ca2 ⁺And Mg ²⁺) washing 2 times, add 0.2ml cell breakdown damping fluid, then incubation 5 minutes at room temperature makes cell separate with the bottom.After the separation, stop suction nozzle (1000 μ l) pressure-vaccum repeatedly, cell is further separated with P1000 pipettor and aerosol.About 75% dissociative inoblast is transferred in each hole of 24 hole tissue culture plate, further carry out enlarged culturing, analyze usefulness for subsequent P CR, transfer in the hole of second 24 hole flat board remaining 25%, carry out enlarged culturing, be used for the body-cell neucleus transplanting test at last.When the cell proliferation in the flat board that is containing 75% initiating cell to when converging, DNA isolation from these clones is used for genetic analysis.

The DNA preparation

The method that is used to separate the DNA that uses for genetic analysis is by Laird etc., Nucleic AcidsResearch, and 1991, Volume 19, and the method improvement of No.15 obtains.Especially, in case reached in a kind of specific hole that is cloned in 24 hole flat boards near converging, then sucking-off substratum from this hole washs attached cell 2 times with PBS.Exhaustion PBS is replaced by the 0.2ml damping fluid, and described cell and digest excess protein from DNA to be separated dissociates.This damping fluid is made up of 100mMTris-HCl (pH8.5), 5mM EDTA, 0.2% SDS, 200mMNaCl and 100 μ g/ml Proteinase Ks.24 hole flat boards are put back into reach minimum 3 hours in the incubator for tissue culture, thereby DNA is discharged and digesting protein.The sticky product of this method is transferred in the 1.5ml Eppendorf tube, added the 0.2ml Virahol with deposit D NA.By centrifugal recovery precipitation, precipitate with 70% ethanol rinsing DNA, air-dry after, precipitation is resuspended in 25-50 μ l contains 10mM Tris, in the damping fluid of pH8 and 1mM EDTA.The pcr analysis that this DNA is used to clone.

Colony screening

Come screening and cloning with two kinds of diverse ways, these two kinds of methods all adopt polymerase chain reaction (PCR).All methods of describing in these chapters and sections all are applicable to the target practice of any other gene, and difference is only for being used for the primer sequence of genetic analysis.

According to first method, with independently the increase product of stable transfection of two pairs of different primers.Detect the existence of targeting vector in the cloned genes group with a pair of primer, and do not consider integration site.Primer is designed to be annealed on the dna sequence dna that is present in the targeting vector.The intensity of the product of this PCR reaction may be relevant with the copy number that is incorporated into the targeting vector in the genome.Therefore, the cell that only contains the targeting vector of a copy by described PCR reaction often produces the more weak band of intensity.Another is designed only to detect the copy that is incorporated into the carrier in the required locus to primer.In this case, a kind of primer is designed to be annealed in the targeting vector, and on the sequence that another kind of primer is designed to be annealed to is non-existent in the targeting vector, air exercise target gene seat is special.In this case, have only when non-existent described site takes place to integrate in the targeting vector next-door neighbour targeting vector, just can detect the PCR product, the target practice incident of indicative of desired.If detect product, then carry out nuclear transplantation with this clone.

Heavy chain for neomycin resistance knocks out construct, usefulness primer Neol (5 '-CTT GAAGAC GAA AGG GCC TCG TGA TACGCC-3 '; SEQ ID NO:42) and IN2521 (5 '-CTG AGA CTT CCT TTC ACC CTC CAG GCACCG-3 '; SEQ ID NO:43) detects the existence of targeting vector in the cell, do not consider the position of integrating.With primer Neol and OUT3570 (5 '-CGA TGA ATG CCC CAT TTC ACC CAAGTC TGTC-3; SEQ ID NO:44) come only to increase specifically those copies of the target practice construct that is incorporated into μ heavy chain gene seat.

Knock out these PCR reactions of the integration of construct for the heavy chain that is used for analyzing described neomycin resistance, use Qiagen PCR test kit.This PCR reaction mixture contains the various primers of 1pmol, 10 times of reaction buffers of 5 μ l, 10 μ l Q solution, 5 μ l DNA and 1 μ l dNTP solution.Water makes the cumulative volume of reaction mixture reach 50 μ l.Under 94 ℃, utilize initial sex change incubation to carry out pcr amplification in 3 minutes.Then, carried out 30 round-robin sex change, annealing and amplifications in 2 minutes at 94 ℃ of 45 seconds of following incubation, 60 ℃ of following 45 seconds of incubation and 72 ℃ of following incubations.Then, with reaction mixture 72 ℃ of warm in nature educating 5 minutes, and at 4 ℃ of following incubations up to from the PCR instrument, taking out mixture.

In alternative method, the locus that adopts one primer cover to increase and practice shooting, and the size of PCR product can be used for determining whether correct target practice.Design a kind of primer and be annealed on the locus zone that is not present in the targeting vector, be annealed to and be present in targeting vector but be not present on the site in the wild type gene seat and design another kind of primer.In this case, can not detect be incorporated into locus do not expect targeting vector on the site.Because the zone of being deleted by targeting vector is different with the drug screening mark that inserts this position in size, the size of product depend on the locus that is increased be the wild type gene type or the target gene type.From contain incorrect insertion or do not insert the clone's of targeting vector DNA cloning at all, produce the single PCR product of the wild type gene seat of expection size.Produce two kinds of PCR products from the allelic cloned DNA amplification that contains correct target practice (" knocking out "), a kind of amplification of representing wild-type allele, a kind of representative since the drug resistance marker replace due to some sequence in the wild-type allele, foreseeable, the reformed allelic amplification of size, allelic length is different from the sequence that is replaced.

Heavy chain for the tetracycline resistance knocks out construct, the short end of use primer (5 '-CTGAGC CAA GCA GTG GCC CCGAG-3 '; SEQ ID NO:45) and long end (5 '-GGG CTG AGA CTG GGT GAA CAG AAG GG-3 '; SEQ ID NO:46).This is to primer amplification wild-type heavy chain gene seat and by the correct locus of practicing shooting of tetracycline construct.Difference between two stripe size is about 0.7Kb.Exist short bar carrying means correctly to practice shooting.

Knock out the PCR reaction of the integration of construct for the heavy chain that is used to analyze the tetracycline resistance, adopt Promega Master Mix test kit.Described PCR reaction mixture contains the various primers of 1pmol, 2.5 μ l DNA and 25 μ l 2X Promega Master Mix.Water makes the cumulative volume of reaction mixture reach 50 μ l.Carried out pcr amplification in 2 minutes 94 ℃ of down initial sex change insulations.Then, under 94 ℃, be incubated for 45 seconds, be incubated for 45 seconds down and be incubated 2 minutes down, carry out 30 round-robin sex change, annealing and amplifications at 72 ℃ at 60 ℃.Then, reaction mixture is incubated 5 minutes down at 72 ℃, and is incubated up to from the PCR instrument, taking out mixture down at 4 ℃.

First round nuclear transplantation

The inoblast that selected wherein immunoglobulin gene is inactivated is used to carry out nuclear transplantation as described in embodiment 2, to produce transgenic ungulates, it contains sudden change and contains the different clock immunoglobulin gene of encoding in endogenous immunoglobulin genes HAC.Alternately, adopt ordinary method to carry out nuclear transplantation, to be inserted in the non-nucleus egg mother cell from the fibroblastic nucleus of selected transgenosis or chromatin (the one or more karyomit(e)s that promptly do not have film bag quilt) and (be described in U.S.S.N.60/258,151) as application on December 22nd, 2000.These methods can also be used for the cell that wherein α-(1,3)-galactosyltransferase, Protein virus and/or J chain nucleic acid are suddenlyd change.

Second takes turns mutagenesis and nuclear transplantation

If desired, from the transgenic ungulates of producing by first round nuclear transplantation, obtain cell (for example, fetal fibroblast).Can carry out another as mentioned above and take turns second allelotrope that gene targeting comes the gene that deactivation is inactivated in practicing shooting in the first round.Alternately, another immunoglobulin (Ig) (for example μ heavy chain, lambda light chain, K light chain or J chain), α-(1,3)-galactosyltransferase or prion gene are taken turns in the target practice at this and are inactivated.Take turns target practice for second, can use the microbiotic of high density, perhaps use the construct that knocks out with different antibiotics resistance marks.Select the antibiotics resistance cell as mentioned above.Selected cell can be used for second as mentioned above and take turns nuclear transplantation, to produce the transgenic ungulates that for example contains two sudden changes and contain the HAC of coding heteroimmunity globulin gene in endogenous immunoglobulin genes.Alternately, the antibiotics resistance cell selected of treatment of selected at first, so that the cell of separation G1 phase as described below, this cell is used to second and takes turns nuclear transplantation.

In order to separate the G1 phase cell that is used for nuclear transplantation, in separation preceding 24 hours, with 5.0 * 10 ⁵Individual cell is taped against on the tissue culture plate of the 100mm that contains 10ml α-MEM+FCS.At second day, dull and stereotyped with the PBS washing, and changed substratum in the 1-2 before separation hour.Go up vibration 30-60 second at Vortex-Genie 2 (Fisher Scientific, Houston, TX, middling speed) then, remove substratum, with 1000G centrifugal 5 minutes, precipitation is resuspended among the 250 μ lMEM+FCS.Select new splitted cell doublet then by the cytoplasmic bridge connection, early stage because these cells are in G1.This separation method is called " shaking off (shake-off) " method.

Embodiment 4: the additive method of the endogenous immunoglobulin genes that is used to suddenly change

In some embodiments of the inventive method, in prion gene, has production heteroimmunity sphaeroprotein in the ungulate of one or more sudden changes, basically by being used in combination homologous recombination technique, importing artificial chromosome, nuclear transplantation and the administration of antibodies of carrying complete xenogenesis Ig locus and eliminate endogenous antibody and realize.More especially, though can also there not be the production that realizes heteroantibody in the animal that suddenlys change in Ig nucleic acid, this method preferably includes target and destroys one or two allelotrope of IgM heavy chain gene and one or two allelotrope of the Ig light chain gene of choosing wantonly.Can pass through the successive homologous recombination, carry out another mating operation then, realize gene knockout.In preferred embodiments, this destroys and realizes by adopting suitable homologous recombination vector at first an allelotrope of the IgM heavy chain gene of the male or female ungulate (for example ox) in the tissue culture to be carried out target.With respect to other somatocyte, it is preferred using fetal fibroblast because these cells breed easily, and easily in tissue culture by genetic manipulation.But, for the present invention, use fetal fibroblast dispensable, in fact, can substitute, and have suitable effect with other clone.

Certainly, this method must make up the DNA construct that has with target practice IgM heavy chain allelotrope homologous zone, makes the construct that is incorporated in the genomic IgM heavy chain of the ungulate allelotrope destroy its expression.Be used for implementing the allelic exemplary carrier of this target practice destruction IgM and be described in following embodiment.In this, being used for providing the method for the carrier construction of homologous recombination on the target practice site is known to those skilled in the art.In addition, in this case, within the level that is structured in those skilled in the art of suitable carrier, be (to vide infra) under the known situation particularly in the sequence of Niu Chonglian and Ig lambda light chain gene, and from the immunoglobulin gene sequence of other ungulates.Homologous recombination for convenience, the carrier that is used to realize homologous recombination and IgM inactivation of gene comprise the DNA part that has in fact with the sequence identity of ungulate IgM heavy chain and Ig light chain gene respectively.Preferably, these have at least 98% sequence identity, are more preferably the sequence of at least 99% sequence identity and higher sequence identity, will be isogenic with the target gene seat, to make things convenient for deletion of homologous recombination and target or deactivation.

Typically and preferably, the homologous recombination body that provides expectation will be provided this construct, the inoblast marker gene of screening for example, and wherein IgM heavy chain gene and/or Ig light chain gene are destroyed effectively.Wherein, exemplary marker gene comprises antibiotics resistance mark, drug resistance marker and green fluorescent protein.Preferred construct is shown among the accompanying drawing 2A, and the parent material that is used for preparing this construct is shown in accompanying drawing 3A and 3B.The Protocols in Molecular Biology of employing standard can be produced and contain two other constructs with endogenous immunoglobulin genes homologous zone, and be applied in the method for the present invention, the side of this immunoglobulin gene is the positive selectable marker (for example antibiotics resistance gene) that is operably connected on the promotor.

The μ that is shown among accompanying drawing 2A and the 3C knocks out two exons that construct is designed to remove the exon of the coding B-IgG CH that is called " C-μ exons 1-4 " and is called the coding membrane spaning domain of " TM exon ".

In order to make up this carrier, will be from the XbaI-XhoI fragment of genome μ heavy chain Niu Xulie, the zone that is called " 1 ", subclone is to commercial dna vector pBluescript (Stratagene, LaJolla, California) in, pBluescript has been done shearing in advance with enzyme XbaI and XhoI.In case the clone obtains this fragment, on contiguous XbaI site, there is NotI Restriction Enzyme recognition sequence, be used to insert the NotI fragment of about 3.5Kb.This fragment contains the neomycin resistance mark, will further describe hereinafter.If desired, can adopt genome μ sequence of heavy chain from another kind of ungulate kind, kind or kind (for example from SwissBull/Holstein cross-fertilize seed μ sequence of heavy chain) with Genbank accession number U63637 preservation.

In case fragment " 1 " is connected among the pBluescript with the neomycin resistance mark, still there is the SacI site of contiguous neomycin resistance mark.With SacI linearizing novel constructs, mend the flat sticky end that produces by SacI digestion with archaeal dna polymerase, become flush end.

The fragment that is called " 2 " is separated with the XhoI-BstI1071 fragment, mends the flat sticky end that is produced by the XhoI-BstI1071 enzymic digestion with archaeal dna polymerase, becomes flush end.

In case finish, final construct contains zone 2, neomycin resistance mark and zone 1 respectively.

For the fibroblastic transfection of ox, digest this construct (in two KpnI site diagrammatic sketch) with Restriction Enzyme KpnI, and this dna fragmentation is used to homologous recombination.

Following assembling neomycin resistance construct.The construct (Katoh etc., Cell Struct.Funct.12:575,1987 that are called " pSTneoB "; Japanese Collection of ResearchBiologicals (JCRB) deposit number VE039) be designed to contain neomycin resistance gene, it is regulated and control by SV40 promotor and the TK enhanser that is positioned at upstream of coding region.The downstream of coding region is a SV40 terminator sequence.The Neo box is cut out from " pSTneoB " as the XhoI fragment.With conventional molecular biology method with segmental end modified be flush end after, with the fragment cloning of flush end to the EcoRV site of carrier pBS246 (Gibco/Life Technologies).The both sides in this site are the loxP sites.The novel constructs that is called " pLoxP-STNeoR " is used to produce μ knockout dna construct.The segmental both sides of the expectation of this construct are loxP site and NotI site, and these two sites are present in the pBS246 cloning vector at first.The NotI fragment of expectation contains loxP-neo-loxP, is used for substituting the μ constant region exon of immunoglobulin (Ig).The SV40 promotor that is operably connected on the neomycin resistance gene activates transcribing of neomycin resistance gene, makes the NotI fragment of wherein expectation replace the cell of the μ constant region that will select according to the antibiotics resistance of its generation.

Obtaining wherein IgM heavy chain allelotrope by after the destructive clone effectively, the donor that this clone is used as nuclear transplantation is produced clone's ungulate fetus (for example Ke Long ox fetus), even one of IgM heavy chain allelotrope ruined fetus or animal.After this, realize that with deriving from for example fibroblastic somatocyte second gene targeting of taking turns destroys, in order to produce wherein second cell that IgM heavy chain allelotrope is inactivated, but adopt the similar substrates that contains different selective markers.

Preferably, parallel with first target practice gene disruption, the somatocyte system of second ungulate (for example ox) is also by genetic modification, and these clones are male source or female source equally.If operated first clone is male, preferably be used for modifying female cell system; Vice versa, if operated first clone is female, then preferably is used for selecting a male sex cell system.In addition, preferably, operated cell comprises ungulate (for example ox) fetal fibroblast.。

In preferred embodiments, female fetal fibroblast is imported a kind of target practice with an allelotrope to the Ig lambda light chain gene and destroys by genetic modification.Similarly, this method adopt have with ungulate (for example ox) but the carrier of Ig lambda light chain homologous zone and selective marker is realized, this DNA construct is designed to come when integration taking place and during with endogenous Ig light chain generation homologous recombination, makes the Ig lambda light chain gene destroyed (deactivation) of target.

In case selected to have the female fibroblast of target practice destructive of expectation, this clone is used as donorcells equally and carries out nuclear transplantation, perhaps is used as the donor of nuclear transplantation from the DNA of this clone.

Alternately, but use be used to destroy first allelic construct similarly, but the DNA construct that contains different selective markers this cell carried out second take turns homologous recombination, come the allelotrope of second Ig lambda light chain of deactivation.

Be used to implement nuclear transplantation, reported especially for the method for producing clened cows and cloned, transgenic ox, and be described in United States Patent (USP) 5,945, in 577.Alternately, can adopt method (being referred to as the Roslin method) in any one that is disclosed in PCT open W095/16670, W096/07732, W097/07669 or W097/07668.The Roslin method is different from the method for University of Massachusetts, employing be the donorcells of stationary phase, rather than outgrowth donorcells.All these patents are incorporated herein by reference fully at this.These nuclear transplantation methods are produced genetically modified clone fetus, and this fetus is used to produce clone's transgenic cattle offspring, at least one allelotrope that for example comprises Ig light chain gene and/or IgM gene destructive offspring that practiced shooting.After having set up this clone, they are used to produce male and female heavy chain and the light chain hemizygote knocks out the fetus and the offspring of (M and F Hemi H/L).In addition, these technology are not limited to be used to produce transgenic cattle, and above-mentioned technology can be used to the nuclear transplantation of other ungulates.

After nuclear transplantation, can take turns the animal that gene targeting comes prestige production phase by the mating ungulate or by carry out second with previously described homology targeting vector.

As previously pointed out, another object of the present invention relates to the male and female heavy chain of generation and the light chain hemizygote knocks out, and it is to adopt the clone of having described to produce that wherein this hemizygote knocks out.This can realize by the offspring that mating is produced according to the method described above, wherein comprises the ruined offspring of allelotrope and the ruined another kind of offspring's mating of allelotrope that comprises the Ig light chain of IgM heavy chain gene.Alternately, this can take turns gene targeting by second by the cell that operation derives from according to the method described above the offspring who produces and realizes.This comprises by the allelotrope of homologous recombination target practice destruction IgM heavy chain gene or the allelotrope of Ig light chain realizes.Comprising after clone that male and female heavy chain and light chain hemizygote knock out (M and F Hemi H/L) produced, this clone is used to produce and comprises this fetus that knocks out or calf.As noted, this can take turns gene targeting by mating or second and realizes.

Knock out after the body in case obtain male and female heavy chain and light chain hemizygote, be used to from the cells of these animals that producing isozygotys knocks out the fetus of (Homo H/L).In addition, this realizes by consecutive gene target practice or mating.In fact, if realize by mating, this will comprise that male heavy chain of mating and light chain hemizygote knock out body and female heavy chain and light chain hemizygote and knock out body, and select to comprise to isozygoty to knock out the offspring of body.Alternately, can in tissue culture, operate from above-mentioned narrowing and knock out the cell of body, to knock out another allelotrope of IgM or Ig light chain (λ) gene.Preferably take turns gene targeting and carry out mating second because this can produce result more fast, particularly because ungulate for example Gestation period of ox long relatively.

Knock out continuously and/or breed in the strategy this, the any donorcells that generates in this process (for example, the donorcells that contains or do not contain sudden change and contain or do not contain xenogenesis Ig nucleic acid in endogenous Ig nucleic acid) in, one or two allelotrope of prion gene is suddenlyd change.Therefore, ungulate with method production described here, on prion gene, Ig heavy chain gene, Ig light chain gene, α-(1,3)-galactosyltransferase and/or J chain, have the sudden change of any amount expectation, and have one or more xenogenesis Ig and/or J chain gene.

The knockout technique of the transgenic ungulates of production expressing human Ig

Be used for producing the method summary of Homo H/L fetus or calf at accompanying drawing 1.Three kinds of schemes have wherein been summarized.First method depends in regenerated fetal cell system and knocks out continuously.This method is the most difficult technically, and risk is the highest, but as above-mentioned, this method may obtain the result quickly than offspring breeding method.Other two kinds of schemes depend on breeds animal.In second kind of scheme, only need in male and female cell system, to carry out respectively knocking out of heavy chain and light chain gene.This scheme does not rely on clone regeneration, is the simplest technical method, need the longest time but finish.Scheme 3 is between scheme 1 and 2.In all schemes, H/L knocks out survival of calf and the difficulty of keeping because potential isozygotys, and has only produced the H/L fetus of isozygotying.If desired, can adopt passive immunotherapy to strengthen the survival that the H/L that isozygotys knocks out calf.

Test design

The present invention preferably relates to and produces that the male heavy chain of hemizygote knocks out body (M Hemi H) and the female light chain of hemizygote knocks out body (F Hemi L), and from these deletion bodies of practicing shooting 40 days fetus of production.Results are from embryo's cell, an allelotrope of the light chain gene seat in the M Hemi H cell is practiced shooting, and an allelotrope of the heavy chain gene seat in the F Hemi L cell is practiced shooting, and generates the cell (Hemi H/L) that H and L locus are deleted by hemizygote.These cells are used to produce 40 days fetus, are separated into fibrocyte from this fetus.

With other H chain allelotrope M Hemi H/L inoblast of practicing shooting, producing MHomo H/Hemi L, and with other L chain allelotrope F Hemi H/L that practices shooting, with generation F Homo L/Hemi H.In order to produce the deletion of isozygotying, drive homozygote with higher drug level and practice shooting.But, may be because this method is difficult to successfully, and need breed.Depending on the exemplary policy that the cre/lox that selects box is practiced shooting allows identical selective system is adopted in more than one target practice deletion.These inoblasts are cloned, collected 40 days fetus, and be separated into fibrocyte.To practice shooting from this clone's fetal cell, H or the deletion of L locus so that generation is isozygotied generate M Homo H/L and F Homo H/L fetal fibroblast.These inoblasts are cloned, produced 40 days fetus, and be separated into fibrocyte.Then Homo H/L fetal fibroblast is used in conjunction with HAC, randomly adopts offspring breeding method.。

Library construction

Make up genomic library with fetal fibroblast.Though it is important having reported the target practice construct homogenic with the cell that is used to clone, it is optional for the present invention.For example, isogenic, isogenic in fact or non-isogenic construct is used to produce sudden change in endogenous immunoglobulin genes.In a kind of possible method, use the Holstein ox, compare with other cattle breeds, in heredity, it has high inbreeding level.In the immunoglobulin gene of different animals, we do not detect any polymorphism.This shows that sequence homology should be very high, and to practice shooting with non-isogenic construct should be successful.

Make up the library by a male sex cell system and a female cell system, carry out " clone property " simultaneously and detect.Can expect, after this method finishes, will prepare a library, and detect a large amount of different fetal cells and be, wherein select a clone to be used to clone purposes as the best.

Make up genomic library with separating from the macromolecule DNA of fetal fibroblast.Classification DNA on the size is inserted into the macromolecule DNA between the 20-23Kb among λShi Juntizaiti λ Zap or the λ Fix.The inventor has obtained very big success with the library of Stratagene preparation.Therefore, DNA isolation and the DNA that will select by size are sent to Stratagene, carry out the library preparation.In order to separate the clone who contains Niu Chonglian and light chain, use radiolabeled IgMcDNA and radiolabeled light chain cdna.In addition, under the situation of needs deletion locus, separate light chain gene group clone.Screen the clone who contains Niu Chonglian and light chain in each fetal cell library.Can expect, screen about 10 ⁵-10 ⁶Individual plaque will be separated to the clone who contains heavy chain or light chain gene seat.In case separated, two locus are cloned among the pBluescript, and carry out restricted mapping.The restriction map of these locus in the Holstein ox is shown in (J Immunol 140:3654-3659 such as Knight, 1988) among the accompanying drawing 2B.In addition, the preparation from the clone who is obtained collection of illustrative plates, be used to assemble the target practice construct.

The production of target practice construct

In case separated heavy chain and light chain, just carried out the preparation of construct.Membrane structure territory by deletion IgM constant region prepares the IgM construct.Shown in Rajewsky and colleague thereof, in mouse, the membrane structure territory of deletion IgM can cause the cytocerastic blocking-up of B, and this is because surperficial IgM is the required signal (Kitamura etc., Nature 350:423-426) of continuity B cell development.The IgM ox of therefore, isozygotying lacks the B cell.Owing in this programme, do not need to lack the newborn infant of the animal of functional Ig, therefore, lack the B cell and can not bring problem.But, if desired, can adopt passive immunotherapy to strengthen the survival of animal up to the final step that imports people Ig locus.

Be used for realizing that knocking out the allelic exemplary target practice construct of IgM heavy chain is shown in accompanying drawing 2A.For heavy chain, with both sides the Xin Meisu box replacements in lox P site with film IgM structure city.Connected membrane structure territory with the neo box by montage, make this membrane structure territory have just in 5 of lox P site ' end has the TAG terminator codon of insertion, so that this membrane structure territory is inactivated.This is placed on 5 ' end of the target practice construct of the 3 ' chromosomal DNA that contains the 5-6kb that has an appointment.

If improve second allelotrope that drug level fails to delete IgM heavy chain or light chain, then adopt cre/lox system (at Sauer, 1998, Methods 14:381-392 is being commented) but the selective marker of deletion.As mentioned below, but the cre/lox system can target deletion selective marker.Delete flag if desired, but then all selective markers are connected with loxP sequence side, with convenient these marks of deletion.

The light chain construct contains ox λ chain constant region (for example being present in lambda light chain constant region among the GenBank accession number AF396698 or the lambda light chain constant region in any other ungulate) and both sides are the puromycin resistance gene boxes in lox P site, and will be that the tetracycline box in lox P site is replaced cow genome with both sides.The DNA of about 5-6kb of lambda light chain constant region gene 3 ' end will be replaced by puromycin resistance gene at 3 ' end.If desired, all carry lox P site at 5 of puromycin resistance gene ' end and 3 ' end, to delete.Because the high homology between the ungulate antibody gene, ox lambda light chain sequence in GenBank accession number AF396698 be expected with from the genome lambda light chain sequence hybridizations of various ungulates, thereby can be used to separate in the ordinary method lambda light chain gene group sequences of various ungulates.These genome sequences can be used in the ordinary method, for example described here those, knock out construct with production and come endogenous lambda light chain in any ungulate of deactivation.

Similarly, can adopt ox κ sequence of light chain among the accompanying drawing 3G or any other ungulate K sequence of light chain to make up the κ light chain and knock out construct.These ox K sequence of light chain can be used as hybridization probe and separate genome K sequence of light chain from various ungulates.These genome sequences can be used in the ordinary method, for example described here those, knock out construct with production and come endogenous κ light chain in any ungulate of deactivation.

Other ungulate genes are selectively suddenlyd change or deactivation.For example, the Ig J chain of endogenous ungulate is knocked out, and is applied to the potential antigenicity of the ungulate Ig J chain in people's the antibody of the present invention with prevention.For the structure of targeting vector, can use the cDNA sequence in the ox IgJ chain zone that is present among the Genbank accession number U02301.This cDNA sequence is used as probe and comes from the BAC library for example RPC1-42 (BACPAC in Oakland, CA) the middle genome sequence that separates the genome sequence of ox Ig J chain or separate the J chain from any other ungulate.In addition, people J chain encoding sequence is directed in the ungulate of the present invention, with functionally expressing human IgA and IgM molecule.The cDNA of these people J chains can obtain from GenBank accession number AH002836, M12759 and M12378.Adopt ordinary method, for example described here those, these sequences are inserted in the inoblast of ungulate fetus.For example, with HAC, yac vector, BAC carrier, cosmid vector or knock out people J chain nucleic acid in the construct and be incorporated in the endogenous Mammals karyomit(e) or maintain independently in the endogenous ungulate karyomit(e).The transgenic ungulates cell that generates is used in the nuclear transplantation method described here, and to produce the ungulate of expectation, it has the heterologous nucleic acid that reduces or eliminate the sudden change of functional ungulate J chain expression and contain expressing human J chain.

In addition, with ungulate α-(1,3)-galactosyltransferase transgenation, to reduce or to eliminate the expression of galactosyl (α 1,3) semi-lactosi epi-position, this epi-position is by α-(1,3)-galactosyltransferase production.If modified by this sugar epi-position by people's antibody that ungulate of the present invention produces, when being applied to man-hour as therapeutical agent, these glycosylated antibody are by in the acceptor and antibody deactivation or elimination sugared epi-position reaction.In order to eliminate these possible immune responses to sugared epi-position, the sequence of ox α-(1,3)-galactosyltransferase gene is used to design and knocks out construct and come this gene (Genbank accession number J04989 in the deactivation ungulate; Joziasse etc., J.Biol.Chem.264:14290-14297,1989).Be disclosed in United States Patent (USP) 6,153,428 and 5,821, these Niu Xulie in 117 or the α of pig-(1,3)-galactosyltransferase sequence can be used to obtain genome α-(1,3)-galactosyltransferase sequence from various ungulates, other ungulates that are lowered or are eliminated with the expression of producing galactosyl (α 1,3) semi-lactosi epi-position.

If desired, the ungulate prion gene is suddenlyd change or deactivation reduces the potential risk of infection, for example mad cow disease (BSE).For the structure of targeting vector, can use the genomic dna sequence (GenBank accession number AJ298878) of bovine prion protein gene, as described in Example 1.Alternately, this genome Protein virus sequence can be used to isolated genes histone virus sequence from other ungulates.Prion gene can come deactivation with method described here.Alternately, previously described being used for can improve according to method described here in the suddenly change method (Denning etc., Nature Biotech., 19:559-562,2001) of α-(1,3)-galactosyltransferase gene or Protein virus of sheep.For example, can adopt described herely be used for cultivating donorcells (for example fetal fibroblast), produce knockout carrier, with knockout carrier transfection donorcells (for example transfection when having spermidine) and/or will be from the transfer of genetic material that knocks out cell improving one's methods to the ovocyte that is used to produce transgenic ungulates.

,, but be necessary to assemble a new target practice construct that contains the difference selective marker for second allelotrope of each locus of practicing shooting if but first selective marker still is present in the cell.Described in table 5, various selection strategies are available and can compare them, and select suitable screening system.At first, by improving drug level (for example, drug level being doubled) second allelotrope of practicing shooting.If unsuccessful, can use a new target practice construct.

Table 5: but selective marker and the medicine that is used to screen

Genomic medicine

Neo ^r G418

The Hph hygromycin B

The Puro tetracycline

The Ecogpt mycophenolic acid

The Bsr blasticidin S

HisD histamine alcohol

The DT-A diphtheria toxin

Adopt the whole bag of tricks, other above-mentioned sudden changes or inactivation of gene can be incorporated in the ungulate of the present invention.In case produced transgenic ungulates clone, adopted hybridization that these additional mutations are incorporated in the ungulate of the present invention with various expectation sudden changes.Alternately, the fetal fibroblast with these additional mutations can be used as the parent material that knocks out endogenous Ig gene and/or import xenogenesis Ig gene.In addition, in endogenous Ig gene, has the parent material that the fetal fibroblast that knocks out the sudden change and/or contain xenogenesis Ig gene is used as these additional mutations or deactivation.

The target deletion of Ig locus

The target practice construct is imported in the fetal fibroblast, for example, pass through electroporation.By using suitable microbiotic, selected to mix the cell of targeting vector.Select to cultivate the clone that drug screening is had resistance.Then, with guanine these clones are carried out feminine gender and select, guanine will be selected those clones of suitably having been integrated.Alternately, be chosen in the clone who survives in the drug screening by PCR.Estimation may need to screen at least, and 500-1000 clone seeks the clone who is suitably practiced shooting.The inventor's estimation is based on Kitamura (Kitamura etc., Nature 350:423-426,1991), and Kitamura etc. find that about 1 quilt is correctly practiced shooting in 300 neo resistance clones when the membrane structure territory of target practice IgM CH.Therefore, suggestion will be in 96 hole flat boards, one group the clone are compiled with 10 clones, and 10 clones' set are screened select the clone that practiced shooting.In case the positive of being accredited as is screened isolating monospecific polyclonal from the clone who compiles.This method can be identified the clone who is practiced shooting.

Because inoblast can be moved, therefore, when generation surpasses about 10 clone in 1 culture dish, just be difficult to distinguish single clone in the process of cultivating.In addition, the clone that should develop efficient transfection breeds strategy.Can use the strategy of various reasonable, for example the dilution clone.

Cre/Lox excises the drug resistance marker

As implied above, but exemplary target practice construct contains the selective marker of being surrounded by side, loxP site, conveniently to use the effective delete flag of cre/lox system.By electroporation, carry the fetal fibroblast of targeting vector with the plasmid transfection that contains Cre.Can use a kind of Cre plasmid of nearest description, it contains GFPcre fusion gene (Gagneten etc., Nucleic Acids Res.25:3326-31,1997).This makes can select to contain proteic all clones of Cre apace.These cells can be selected by the FACS sorting or by the manual green fluorescence cell of collecting of micrurgy.Green cell is considered to carry active Cre recombinase of transcribing, and has therefore deleted the drug resistance marker.Will be selected as the Cre cell clone of expressing, and clone by pcr analysis and to detect the drug resistance marker.Those are determined the cell of having sheared are grown to little clone, separate, and in selecting substratum, detect an aliquots containig, deleted to determine drug resistance gene.Other aliquots containig is used to the target practice deletion of next round.

Use the tactful immunoglobulin gene that changes in other ungulates of practicing shooting

In order to change the immunoglobulin gene of other ungulates, targeting vector is designed to contain three main region.First zone and the locus homology of being practiced shooting.Second zone is the drug screening mark, and it is specifically replaced by the part of target practice locus.The 3rd zone is similar to first zone, and by the locus homology of being practiced shooting, but in the wild-type genome, its not with first region adjacent.Homologous recombination between the wild type gene seat of targeting vector and expectation makes in the locus sequence that shows as on the targeting vector between two zones of homologous deleted, and replaces this sequence with the medicine resistance marker.In preferred embodiments, total size in two homology zones is about 6kb, and the size in second zone of the part of the locus that replacement is practiced shooting is about 2kb.This strategy of practicing shooting is widely used in the interior kind from prokaryotic cell prokaryocyte to people's cell on a large scale.The uniqueness of each carrier is to be selected for the locus of gene targeting method and used sequence in those strategies.This method can be used to various ungulates, includes, but are not limited to goat (Capra hircus), sheep (Ovisaries) and pig (Sus scrufa) and ox (Bos taurus)

Be used for also can being widely used in ungulate at the electroporation of the cell target practice specific gene of ungulate.Ordinary method described here is changeable, the sudden change of practicing shooting is imported in the genome of other ungulates being suitable for.Can make amendment to electroporation conditions (voltage and capacity), to optimize the quantity of the transformant that from other ungulates, obtains.

In addition, the strategy (that is, removing all encode exon and intervening sequences with the carrier that contains the regional homologous zone that directly is connected with removed exon side) that is used herein to target practice ox heavy chain gene seat also can similarly be applied to other ungulates.For example; on the immunoglobulin heavy chain gene seat of sheep (Ovisaries), carry out the analysis of extension sequence; on structure and sequence, immunogene seat highly similar to the cow genome seat (GenBank accession number Z71572, Z49180-Z49188, M60441, M60440, AF172659-AF172703).Except be used to reset the cDNA sequence of public continuous immunoglobulin chain in a large number by report, reported the genome sequence column information of heavy chain gene seat, it comprises heavy chain 5 ' enhanser (GenBank accession number Z98207), 3 ' μ transition zone (Z98680) and 5 ' μ transition zone (Z98681).The complementary mRNA sequence of the secreted form of sheep heavy chain is with accession number X59994 typing.The sequence of this typing contains the complete sequence of four coding exons, they and corresponding ox sequence height homology.

Information about described sheep locus obtains from GenBank, is used for determining and Niu Xulie height homologous zone, for the primer that is designed for pcr analysis.Because use non-isogenic dna that the ox cell is practiced shooting, find to be used as the similar conservative index of sequence possibility between the cattle breeds to sheep sequence height homologous zone.Have similarity between the sequence of the immunoglobulin loci of known ox and sheep and the structure, can expect, can successfully be applied to described sheep system in order to the target practice strategy of removing the B-IgG locus.In addition; existing information about pig (Susscrofa, accession number S42881) and goat (Capra hircus, accession number AF140603) shows; the immunoglobulin loci of these two species is also enough similar to described cow genome seat, therefore can utilize target practice strategy of the present invention.

Embodiment 5: ox IgM knocks out

Following method is used for producing wherein, and an allelotrope of heavy chain immunoglobulin (μ) locus passes through homologous recombination and ruined ox fibroblast.By removing the exons 1-4 of μ locus (corresponding to the IgM heavy chain gene), and replace with the neomycin resistance gene of a copy, generation is used to implement the DNA construct that IgM knocks out.Use this construct, obtained to be successfully used to the neomycin resistance clone of nuclear transplantation, and will be implanted in the receptor cow from the blastaea of this clone.In addition, with PCR method in these blastaeas some are detected, to determine that the target insertion has taken place rightly in the μ locus.Produce blastaea by the nuclear transplantation method from the several clones that obtained, what show gestation is that hemizygote IgM-knocks out fetus.In addition, produced and comprised the male and female cell system that single IgM heavy chain (μ) knocks out.Can expect, will from hematopoietic cell system, carry out mating by clone's animal, will produce wherein two offsprings that the μ copy all is inactivated.As said, the offspring who has sudden change in IgM and prion gene is produced in the ungulate mating that these offsprings can knock out with Protein virus.Alternately, as described in embodiment 1, can be come one or two allelotrope of deactivation prion gene from offspring's cell by genetic modification.These methods will be described hereinafter in further detail.

DNA construct

At the DNA that is used for all conversions described in the presents according to following production.Four main exons (except the membrane spaning domain exon), CH1-4 is connected by the XhoI restriction site of downstream (CH4) end and the side, XabI site of upstream termination (CH1).The construct that is used for transfection method is formed (accompanying drawing 3D and 3E) by the 1.5kb genome sequence in downstream, XhoI site and the 3.1Kb genome sequence of upstream, XabI site.These sequences are as described herein separates from the Holstein ox from the lactation cows of Massachusetts.The neomycin resistance mark is inserted on the fragment that is positioned at these two segmental 3.5Kb, replaces the 2.4Kb DNA that originally contained CH1-4, derives from original gene group sequence.The skeleton of described carrier is pBluescriptII SK+ (Stratagene), the insertion fragment of purifying 8.1Kb, and with its transfection bovine fetal fibroblast, this construct is shown in accompanying drawing 3A-3C.Other μ that contain other homologous regions and/or contain another kind of antibiotics resistance gene knock out construct, and endogenous μ heavy chain gene is used to suddenly change.

Transfection/knockout technique

(CA USA) carries out for Qiagen, Valencia with commercial reagent Superfect Transfection Reagent in the transfection of tire ox.

From the production ox inoblast through the male Charlais ox that disease detects of Hematech ' s Kansas instrument, and deliver to Hematech ' s Worcester Molecular BiologyLabs, be used for described all tests.Any other ungulate kind, kind or kind can be used as the source of these donorcellses (for example somatocyte, newborn fetal fibroblast).Carry out genetic modification for described donorcells, to contain the sudden change that reduces or eliminate functional endogenous Ig expression.

The substratum that is used to cultivate bovine fetal fibroblast is grouped into by following one-tenth: 500ml α MEM (Bio-Whittaker # 12-169F); 50ml foetal calf serum (Hy-Clone#A-1111-D); 2ml microbiotic/anti-mycotic agent (Gibco/BRL # 15245-012); 1.4ml 2 mercapto ethanol (Gibco/BRL # 21985-023); 5.0ml L glutamine (SigmaChemical # G-3126) and 0.5ml tartrate tyrosine (Sigma Chemical # T-6134).

In the day before yesterday of transfection, seed cells in the tissue culture ware of 60mm, by microscopic examination, determine to reach the target degree of converging of 40-80%.

On the same day of transfection, be that the 5 μ gDNA in serum-free antibiotic-free substratum of 150 μ l mix with 20 μ l Superfect transfection reagents with cumulative volume, at room temperature left standstill 5-10 minute, to form the DNA-Superfect mixture.When forming described mixture, remain to remove substratum the fibroblastic 60mm culture dish of ox of transfection from containing, with 4ml phosphate buffer normal saline flushing cell 1 time.The 1ml growth medium is added in the DNA/Superfect mixture of 170 μ l, immediately it is transferred on the cell in the 60mm culture dish.With cell incubation 2.5 hours under 38.5 ℃, 50% carbonic acid gas.After DNA/Superfect mixture incubation, the sucking-off substratum is used 4ml PBS washed cell 4 times at cell.Add the 5ml perfect medium, with culture at 38.5 ℃, 5%CO ₂Under be incubated overnight.With PBS washing 1 time, contain 0.3% tryptic PBS at 37 ℃ of following incubations with 1ml then, then up to determine cell and plate isolation by microscopic examination.The cell branch that derives from each 60mm culture dish is installed in 24 holes of 24 hole tissue culture plate (41.7 μ l/ hole).In each hole, add the 1ml tissue culture medium (TCM), at 38.5 ℃ and 5%CO ₂Dull and stereotyped 24 hours of following incubation.

In all transfections operating periods, carry out false transfection with the Superfect/PBS mixture that does not contain DNA, because can expect and do not contain neomycin resistance gene in those cells, and can expect that all cells is all dead after joining G418 in the tissue culture medium (TCM).This as the cell positive of accepting DNA select negative control.

After 24 hours, the tissue culture medium (TCM) that again 1ml is contained the G418 of 400 μ g adds in each hole at incubation, and the ultimate density that makes G418 is 200 μ g/ml.Cell is put back in the incubator, and the G418 that carried out 7 days selects.During this period, the necrocytosis situation of the dull and stereotyped and false transfection flat board of monitoring transfection, and in 7 days contains seldom or does not contain viable cell from most of holes of false transfection, and the flat board that contains the cell of having accepted described DNA then demonstrates good cell and grows.

After 7 days selection phase, the cell in the hole that will converge from 90-100% contains 0.3% tryptic PBS with 0.2ml and dissociates, and it is transferred on the tissue culture plate of 35mm, to carry out enlarged culturing and incubation, at least 50% converge up to them, at this moment, contain 0.3% tryptic PBS pair cell with 0.6ml and carry out trypsin treatment.On each 35mm tissue culture plate, the 0.3ml in the cell suspending liquid of 0.6ml is transferred to 12.5m ²In the tissue culture flasks, carry out further enlarged culturing.Remaining 0.3ml is inoculated in the culture dish of 35mm, and incubation, minimumly reach about 50% and converge up to them, extract DNA and be used for pcr analysis processing from those dull and stereotyped cells this moment.To be retained in from the flask of each clone in the incubator, analyze through these, and, then can stop cultivating, perhaps be preserved for later nuclear transplantation and freezing preservation if the DNA that they do not contain expectation integrates up to them.

The screening of targeted integration

As mentioned above, the DNA source that is used to screen the transfectant that contains DNA construct is a 35mm tissue culture ware that contains the cell to be analyzed that once goes down to posterity.Be prepared as follows DNA, this DNA is suitable for Laird etc., Nucleic Acids Res., 19:4293) disclosed method.In brief, be prepared as follows DNA.Prepare cell lysis buffer solution with following composition: 100mM Tris-HCl pH of buffer 8.5; 5mM EDTA, pH8.0; 0.2% sodium sodium lauryl sulphate; 200mM NaCl and 100 μ g/ml Proteinase Ks.

Sucking-off substratum from each 35mm tissue culture ware is replaced with the above-mentioned damping fluid of 0.6ml.Culture dish is put back in the incubator kept 3 hours, during this period, cytolysis and protein digestion take place.After this incubation, lysate is transferred in the Eppendorf tube of 1.5ml, and added the different deposit D NA of 0.6ml Virahol.By putting upside down centrifuge tube, centrifuge tube is fully vibrated, be allowed to condition at then and left standstill under the room temperature 3 hours, after this with 13, centrifugal 10 minutes of 000rpm is deposited in the Eppendorf tube DNA and deposits.Discard the supernatant liquor of each centrifuge tube, precipitate 1 time with 70% washing with alcohol.70% ethanol is removed in suction, air-dry DNA precipitation.In case dry, each precipitation is resuspended in 30-50 μ l Tris (10mM)-EDTA (1mM) damping fluid, among the pH7.4, allow its hydration and dissolving spend the night.For each polymerase chain reaction (PCR) program, use the various dna solutions of 5-7 μ l.

With two independently the PCR program analyze transfectant.First program is used two kinds of primers, and described two kinds of primers expection is annealed to the site that all is positioned at the DNA that is used for transfection.The neomycin resistance box homology of first kind of primer sequence and described DNA construct, and second kind of primer sequence produces the PCR product of 0.5Kb apart from its about 0.5Kb.Especially, use primer Neol (5 '-CTT GAA GAC GAA AGG GCC TCG TGA TACGCC-3 '; SEQID NO:42) and IN2521 (5 '-CTG AGA CTT CCT TTC ACC CTC CAGGCACCG-3 '; SEQ ID NO:43).Qiagen PCR test kit is used to this PCR reaction.The PCR reaction mixture contains the various primers of 1pmol, 5 μ l, 10 * reaction buffer, 10 μ l Q solution, 5 μ l DNA and 1 μ l dNTP solution.It is that 50 μ l. employing was carried out this pcr amplification in 2 minutes in 94 ℃ of sex change insulations at first that water makes the cumulative volume of reaction mixture.By under 94 ℃, being incubated for 45 seconds, being incubated for 45 seconds down and being incubated 2 minutes down, carry out 30 round-robin sex change, annealing and amplifications then at 72 ℃ at 60 ℃.Then, allow mixture be incubated 5 minutes down, be incubated up to from the PCR instrument, taking out mixture down at 4 ℃ at 72 ℃.Alternately, can under appropriate reaction conditions, carry out the PCR reaction of a standard, use and described any other primer of regional homologous that knocks out construct that is incorporated in the cellular genome, have resistance owing to having integrated DNA construct with the cell that confirms survival in G418 selects.

Integrate because expection only has the transfectant (μ locus) on desired location of less ratio to contain DNA, therefore, use another that primer is determined, not only the DNA that is imported is present in the genome of described transfectant, and is integrated on the position of expectation.Use is arranged in a kind of primer of neomycin resistance box of described DNA construct and expection only just are annealed to outside the 1.8Kb (being arranged in outside the zone that DNA construct comprised that transfection uses because of homologous region) when described DNA is incorporated on the appropriate location of IgM locus a kind of primer, is used to detect the PCR method of appropriate integration.Design described primer, it is annealed to the dna sequence dna of those sequences that the next-door neighbour represents in the described DNA construct, if will be integrated on the expectation site (be arranged in the zone that there is DNA construct and the dna sequence dna of contiguous locus was before determined with it at genome).Especially, in this is analyzed, use primer Neol and OUT3570 (5 '-CGA TGA ATG CCC CAT TTC ACC CAA GTC TGTC-3; , SEQ ID NO:44).As mentioned above, this PCR reaction uses Qiagen PCR test kit to carry out first PCR reaction, is integrated in the cell to confirm described target practice construct.Alternately, can adopt any proper reaction conditions and any and described be incorporated in the cellular genome other primers of regional homologous that knock out construct and with described cellular genome in integration site upstream or other primers of downstream area homologous, carry out this pcr analysis.

Adopt these methods, at the DNA construct targeted integration in suitable locus, to 135 independently the 35mm flat board screen.Be determined from the DNA of 8 flat boards and contain the DNA construct of appropriately being practiced shooting, therefrom select 3 to be used for the nuclear transplantation method.These clones are named as " 8-1C ", " 5-3C " and " 10-lC ".The residue blastaea that is not used for being transplanted to receptor cow is used to extract DNA, this DNA is carried out extra pcr analysis.This analysis of the primer of screening first of using nested PCR method use also to be used for transfectional cell series is effective.

As mentioned above, produce three kinds of clones with the gene targeting construct that designs the exons 1-4 that removes the μ locus.To use these clones of target insertion male that are after testing of the test of PCR-based to be used for nuclear transplantation.The construct that detects appropriate target by PCR screens the remaining blastaea that produces from those nuclear transplantation.Obtain the positive blastaea of following frequency:

Clone 8-1C:6/8

Clone 10-1C:2/16

Clone 5-3C:0/16

Though in the time of the 40th day, to 11 pregnancies altogether, during by the 60th day, 7 fetuses are dead by ultrasound examination in gestation.Remaining 4 fetuses are through handling producing new fetal fibroblast, and remaining organ is used for production small org sample to carry out pcr analysis.The result who analyzes is as follows:

Clone 8-1C:2 fetus, 1 fetus detects through PCR and is the target insertion positive

Clone 10-1C:1 fetus, detection is that target inserts the positive through PCR

Clone 5-3C:1 fetus, detection is that target inserts feminine gender through PCR

Surprisingly, only be 2/16 though the 10-1C blastaea detects to target inserts the male frequency, pass through the PCR test positive from 60 days fetus of a survival of this clone acquisition.Also obtain positive fetus from 8-1C.DNA to all tissue samples carries out the southern blotting technique analysis, confirms that described construct not only correctly practices shooting (carrying out PCR by the short homologous region that exists in to original construct measures) on an end, and also correctly practices shooting at the other end.Result according to obtaining so far thinks that two heavy chains that produced from two independent integration incidents knock out fetus.In addition, because these fetuses are from two different clones, therefore, at least one fetus may all correctly have been integrated construct on two ends.In case the southern blotting technique analysis confirms that targeting vector is all practiced shooting rightly at two ends, then carries out further nuclear transplantation, produce conceived to other mature fetuses.

Nuclear transplantation and embryo transfer

Carry out nuclear transplantation with K/O clone (8-1-C (18)), generate 8 embryos.At Trans Ova Genetics (＂ TOG ＂; Iowa), in will 6 embryo transfer to 3 anosis acceptors altogether from this batch.

Embryos frozen is transplanted in 10 anosis acceptors, is obtained anosis female fibroblast.After confirming gestation in 35-40 days, formulate the fetus recycle program.

Cyesiognosis and fetus are reclaimed

Transplanted pregnant situation by the ultrasonography inspection from 18 acceptors of the clone embryos that knocks out fetal cell.The result is summarised in the table 6.

Table 6: the gestation when using the μ heavy chain to knock out 40 days of donor western part

Clone ID	The transplant recipient numbering	Gestation (%) in the time of the 40th day
Clone ID	The transplant recipient numbering	Gestation (%) in the time of the 40th day	8-1-OC	5	4(80)
10-1-C	6	4(67)	8-1-OC	5	4(80)
10-1-C	6	4(67)	5-3-C	5	3(60)
Amount to	16	11(69)	5-3-C	5	3(60)

Cyesiognosis

The pregnant situation from 3 acceptors of the clone embryos that knocks out cell (8-1C) has been transplanted in inspection; 1 be opened (open), and other 2 needs were confirmed after 1 month again.

The foundation of fetus recovery and clone

11 pregnant oxen of nourishing the K/O embryo when obtaining the 40th day.In the time of the 60th day, from these oxen, take out 4 fetuses alive.From all 4 fetuses, set up clone, and the freezing preservation of clone is stand-by.In addition, the inventor has collected the tissue samples from described fetus, and it is freezing, delivers to Hematech Molecular Biology Lab and carries out the PCR/DNA engram analysis.

All 4 clones all are male sex cell systems.In order to obtain female cell system, set up clone and freezing preservation the fetus (6) that TransOva Genetics collects in the time of 55 days with the gestation of anosis acceptor foundation gestation, for setting up in the future K/O clone.Recently, one of alleged occurrence contains the female cell system that μ knocks out.This female cell system can be used to produce cloned animal, the animal mating that can allow cloned animal and produce from male sex cell system, and screening contains the offspring that two μ knock out in the offspring.

If desired, come the cell that knocks out fetus or knock out the offspring of self-generating can be used to second and take turns the offspring of nuclear transplantation with the outer clone of delivery capacity.Can be frozen from the cell that knocks out fetus first or knock out the offspring and to form clone, knock out the source of the donorcells of ungulate as producing other.

Embodiment 6: α-1,3-galactosyl shift the active transgenic ungulates that reduces of alcohol

If desired, produce wherein α-1, the transgenic ungulates that the 3-galactosyltransferase is suddenlyd change, the heteroantibody of (1,3)-semi-lactosi epi-position that prevents to have semi-lactosi is by glycosylation.Produce wherein α-1 by homologous recombination, the ox fibroblast that an allelotrope of 3-galactosyltransferase is suddenlyd change.Be used to produce α-1, the DNA construct that the 3-galactosyltransferase knocks out cell is used to puromycin resistance gene (puro, as in this description) and Transcription Termination box (STOP) be inserted in the exon 9 that contains catalyst structure domain, to stop functional total length α-1,3-galactosyltransferase mRNA transcribes.Therefore, the immature α-1 of generation, 3-galactosyltransferase transcript lacks catalyst structure domain.DNA construct (being α-1,3-galactosyltransferase knockout carrier) kind independently in the ox fibroblast, is separated the tetracycline resistance clone by electroporation to three then.According to pcr analysis, in some clones, in exon 9 zones homologous recombination takes place.Therefore, generate wherein α-1, the ox fibroblast that an allelotrope of 3-galactosyltransferase is suddenlyd change.If desired, second allelotrope that can suddenly change with identical knockout carrier, the another kind of knockout carrier that has different antibiotics resistance genes with higher antibiotic concentration or use are selected to isozygoty and are knocked out cell.This method can also be applied to the cell from other ungulates, is used for the transgenic cell of nuclear transplantation method described here with production, produces transgenic ungulates of the present invention.

These methods are further described hereinafter.

α-1, the structure of 3-galactosyltransferase knockout carrier

Following production α-1,3-galactosyltransferase knockout carrier (accompanying drawing 23).In order to separate α-1, genomic dna around the exon 9 of 3-galactosyltransferase gene, with following primer to coming the pcr amplified dna probe: 5 '-gat gat gtc tcc agg atg cc-3 ' (SEQ ID NO:61) and 5 '-gac aag ctt aat atc cgc agg-3 ' (SEQ ID NO:62).Use this probe, cow genome group lambda particles phage library is screened, and identify 7 positive lambda particles phage clones.By restricted mapping, further a clone who contains from the fibroblastic DNA of male Charolais ox is analyzed.The NotI-XhoI genomic fragment subclone that will contain exon 9 inserts puro and STOP expression cassette then on the AviI site of NotI-XhoI genomic fragment in pBluescript II SK (-), this site is at 5 of catalyst structure domain ' end.(DT-A Gibco) also is added in this vector construction body the diphtheria toxin gene, to kill the cell that the expression cassette of wherein practicing shooting is integrated by non-homology ground.

Transfection/knockout technique

As follows, with conventional electroporation scheme, the fibroblast of three fetuses (two kinds from male Jersey ox, and a kind of from female Jersey ox) is carried out transfection.The substratum that is used to cultivate bovine fetal fibroblast contain 500ml α MEM (Gibco, 12561-049), 50ml foetal calf serum (Hy-Clone #; ABL13080), 5ml penicillin-Streptomycin sulphate (SIGMA) and 1ml 2 mercapto ethanol (Gibco/BRL#; 21985-023).In the day before yesterday of transfection, cell is implanted in the T175 tissue culture flasks, have the target degree of converging of the 80-100% that determines by microscopy.On the same day of transfection, with about 10 ⁷Individual ox inoblast Trypsinization, and with α-MEM substratum washing 1 time.After cell being resuspended in 800 μ l α MEM, the DNA of 30 μ g is added in the cell suspending liquid, by the pressure-vaccum thorough mixing.Cell-DNA suspension is transferred in the electroporation sample pool, and, carried out electroporation under 000V and the 50 μ F 1.After this, will be taped against by the cell of electroporation on 20 24-hole flat boards with the α-MEM substratum that has added serum.Cultivate after 48 hours, substratum is replaced with the substratum that contains 1 μ g/ml tetracycline, culturing cell 2-3 week is selected the tetracycline resistant cell.After the selection, select all colonies that converge near 100%, from colony, extract the homologous recombination incident that genomic dna screens by PCR expectation.

Screening to targeted integration

As mentioned above, use PUREGENE DNA separating kit (Gentra Systems),, from each hole in 24 holes, extract genomic dna independently according to manufacturer's scheme.Every kind of genomic dna sample is resuspended among the 10mM Tris-Cl (pH8.0) and 1mMEDTA (EDTA) of 20 μ l.With following primer to screening by PCR: 5 '-aag aag agaaag gta gaa gac ccc aag gac-3 ' (SEQ ID NO:63) and 5 '-cct ggg tat aga caggtg ggt att gtg c-3 ' (SEQ ID NO:64).Article one, the sequence of primer is positioned at α-1, and in the 3-galactosyltransferase knockout carrier, and the sequence of another primer is positioned at outside the integrative vector of the endogenous gene seat of being practiced shooting (accompanying drawing 23) just.Therefore, the PCR product of expectation should only just can detect when described knockout carrier is incorporated in the target gene seat by homologous recombination.

The PCR reaction mixture contains 18.9 μ l water, 3 μ l, 10 * LA PCR damping fluid II (contains Mg ²⁺), 4.8 μ l dNTP mixtures, 10pmol forward primer, 10pmol reverse primer, 1 μ l genomic dna and 0.3 μ l LA Taq.By under the following conditions reaction mixture being incubated: 85 ℃ 3 minutes, 94 ℃ 1 minute, 98 ℃ 10 second and 68 ℃ 15 minutes, carry out 40 round-robin PCR.Behind PCR,, select the tetracycline resistance clone (accompanying drawing 23) of the PCR product that produces the expection size by the electrophoretic analysis reaction mixture.Therefore, successfully produced wherein α-1, the ox fibroblast that an allelotrope in the 3-galactosyltransferase locus is suddenlyd change by knockout carrier.

Embodiment 7: the alternative method of using adeno associated virus sudden change endogenous gene to produce transgenic ungulates

Can replace the target sequence (Inoue etc., Mol.Ther.3:526-530,2001) that exists in the cellular genome specifically with adeno associated virus (AAV); Hirata etc., J.Virol.74:16536-16542,2000); Inoue etc., J.Virol.73:7376-7380,1999); With Russell etc., Nat.Genet.18:325-330,1998)).Compare with more conventional gene targeting method, its gene targeting rate is very effective.AAV has broad host range and has tissue specificity, comprises the specificity to ox and human skin fibroblast.Therefore, can produce at endogenous immunoglobulin (for example μ heavy chain, lambda light chain, K light chain and J chain), α-1, have the transgenic ungulates cell of one or more sudden changes in 3-galactosyltransferase or the prion gene with AAV.Then, these transgenic cells can be used in the nuclear transplantation method described herein, to produce transgenic ungulates of the present invention.

Use AAV, cause B-IgG heavy chain gene seat to be recombinated with the frequency that is higher than the conventional gene targeting strategy of previous usefulness (being electroporation and fat transfection method) and is reached.In first round gene targeting test, in 73 stable transfection that contain the DNA that is imported by the AAV carrier, obtain 5 correct fibroblast clonings of practicing shooting

These tests are carried out according to following.

The AAV knockout carrier

The AAV construct can be by inserting exogenous array or destroying gene with the new sequence replacement endogenous sequence that is present in the AAV carrier simply.Accompanying drawing 24 shows transplants the AAV construct, and wherein 4 codings of all of B-IgG heavy chain μ constant region exon is present on the BamHI-XhoI fragment of 2822bp.To be present in the 1.16Kb fragment that contains the neomycin resistance mark among the commercially available carrier pMClNeo is inserted into and is present in from the SacII site in the exon 4 of the μ heavy chain gene seat of Holstein ox.This locus is the locus that is contained in the phage clone of the separated embodiment of generation 3 described knockout carriers.In order to produce the AAV carrier, the benefit of the SacII site in the μ heavy chain gene seat is flat, make up flush end, subsequently it is connected with flush end SalI joint (New England Biolabs).Then, the XhoI fragment that contains the pMClNeo of neomycin resistance gene is connected to by the SalI joint and adds on the SalI site in the locus.Because XhoI and SalI restriction site have the end that is complementary, and can carry out this connection.This knockout carrier is inserted in the endogenous μ heavy chain gene with causing the destroyed property of neomycin resistance gene, thereby described μ heavy chain gene is inactivated.The generation unit that this gene is inactivated need make the zone disappearance of described endogenous μ heavy chain gene seat.

Design a kind of alternative carrier and during practicing shooting, from the endogenous gene seat, remove exon 3 and 4, cause these two exons to be replaced (accompanying drawing 25) by the neomycin resistance gene of a functional copy.Employing pcr amplification genomic dna from female Jersey ox is produced this construct.Especially, with the following primer 3 ' homologous region that increases: 5 '-GGG GTC TAG Agc agacac tac act gat ggg ccc ttg gtc c-3 ' (SEQ ID NO:65), this primer has added an XbaI restriction site, with 5 '-GGG GAA GCT Tcg tgtccc tgg tcc tgt ctgaca cag-3 ' (SEQ ID NO:66), this primer has added a HindIII restriction site.With following primer amplification 5 ' homologous region, 5 '-GGG GCT CGA Ggt cgg cga agg atg ggggga ggt g-3 ' (SEQ ID NO:67), it has increased an XhoI restriction site, with 5 '-GGG GGG TAC Cgc tgg gct gag ctg ggc aga gtg gg-3 ' (SEQ ID NO:68), it has added a KpnI restriction site.Capitalization Nucleotide in these primer sequences can not be annealed on the μ heavy chain gene seat, adds restriction site in the primer with convenient follow-up subclone step but be included in.Add preceding 4 guanines,, be not positioned at primer real terminal site and inner site because Restriction Enzyme does not cut so that the real end of restriction site with described primer separated.5 ' homology head of district 1.5Kb contains exons 1 and 2.5 ' homologous region also contains preceding 25 Nucleotide of exon 3, to keep the acceptor splicing site of exon.Acceptor splicing site makes exon 3 can be used for montage, thereby prevents that exons 1 and 2 from may form unusual film in conjunction with product by montage in the membrane spaning domain in downstream.3 ' homologous region length is 1.24Kb, contains the zone in next-door neighbour's exon 4 downstreams.

For the construct shown in the accompanying drawing 24, adopt standard method, described target practice expression cassette is inserted in the AAV carrier of length terminal repetition (LTR) sequence that contains virus of (J.Virol.70:1542-1553,1996) reports such as Ryan.(Inoue and Russell, J.Virol.72:7024-7031,1998) as described previously, use the TtetA2 package cell line, with described AAV carrier package in capsid, and according to the previous described purifying (Zolotukhin etc. that carry out, GeneTherapy, 6:973-985,1999).For the construct shown in the accompanying drawing 25, can use aforesaid method or any other standard method, described target practice box is inserted in described AAV carrier such as Ryan or any other AAV carrier (for example deriving from the commercially available carrier of Stratagene), produces the virus that contains described carrier.

Transduction method

To be inoculated into 40,000 cells/well from the inoblast of Jersey ox in the hole of culture plate in 48 holes, in perfect medium, at 38.5 ℃ and 5% CO ₂Following cultivation, up to cell attachment on the lower surface in hole.In case cell attachment is removed substratum, replace to 0.2ml and contain the bright new substratum of the AAV particulate with carrier shown in the accompanying drawing 24, infection multiplicity (MOI) is 500-20,000 particle/cell.The selection of MOI is based on and is determined at colony number and the colony trial test at interval that is produced during the drug screening.The incubation flat board spends the night.Behind the incubation, the PBS rinsing of no calcium and magnesium of the hole that will be transduceed, pancreatin or cell dissociation damping fluid make cell separate with the hole as mentioned above.By the dissociated cell of pressure-vaccum gently, obtain the cell suspending liquid of homogeneous, make the cell in described hole redistribute in 10 100mm tissue culture wares, each culture dish is incubated overnight with perfect medium.

In this 100mm culture dish behind the incubation, substratum is replaced by contains the selection substratum that concentration is the G418 of 350 μ g/ml.Changed in every 2-3 days and select substratum once, on surface, have macroscopic colony at culture dish.At this moment, select single colony, transfer in the container separately.

Mark contains the zone of colony on the outside surface of tissue culture ware.In case iris out all colonies, sucking-off substratum from flat board uses the PBS of no calcium and magnesium to wash dull and stereotyped 3 times.After the washing, the 1 * pancreatin submergence flat board with dilution in 1: 25 at room temperature leaves standstill, and begins to separate with planar surface up to visible colony.Keep dull and stereotyped fixing, float on another dull and stereotyped position to prevent isolating colony.Choose the cell lump that volume is 50 μ l with suction nozzle, the content of suction nozzle is transferred in the hole of 24 hole tissue culture plate.In case all colonies all are transferred, add the perfect medium that contains G418, separated clone is increased, converge to approaching.

When a hole when converging, its PBS with no calcium and enzyme is washed 2 times.Make cellular segregation with 0.2ml cell dissociation damping fluid.20 μ l cell suspending liquids are transferred in the 24 new hole flat boards, and remaining cell is attached on the 24 original hole planar surfaces after adding the 2.0ml perfect medium again.Cultivating 24 original hole flat boards to 100% converges.New flat board is as the source of the appropriate target practice cell that is used for ox cloning process in the future.

When a hole from 24 original hole flat boards reaches 100% when converging, remove substratum, use PBS

Washed cell 1 time.Remove PBS, be replaced by the cell lysis buffer solution of (Nucleic Acids Res.19:4293,1991) employings such as Laird.In brief, the lysis buffer of 0.2ml is added in the hand-hole, this damping fluid contains 200mM NaCl, 100mM Tris-HCl pH8.5,5mM EDTA, 0.2% SDS and 100 μ lg/ml Proteinase Ks.Flat board is put back in the incubator again, reached 3 hours to spending the night.Then, the cytolysate of viscosity is transferred in the Eppendorf tube.Add isopyknic Virahol with deposit D NA.After centrifugal 10 minutes, abandoning supernatant will precipitate and use 0.5ml70% washing with alcohol 1 time in Eppendorf centrifuge.After removing ethanol, air-dry DNA precipitation is resuspended in the 35 μ lTE damping fluids (10mM Tris pH8 and 1mMEDTA).The aliquots containig of 3 μ l is used for pcr analysis.

Pcr analysis

Adopt pcr analysis, the dna sample that the resistance of the AAV particle transduction of using by oneself is cloned screens, and seeks the correct target practice of described carrier.This screening strategy uses a kind of interior primer of DNA that is annealed to coding drug screening mark, and the another kind of primer that is annealed to outside the sequence that still exists in described AAV target practice particle in the target gene seat.Only when AAV target practice DNA is incorporated on the desired location of endogenous gene group, just can detect the PCR product.

What obtain from use the test of these AAV particulate singles target the results are shown in the accompanying drawing 26.Based on this analysis, 5 clones in 73 independent clonings contain the carrier DNA of correct target practice.

This method also can be used with the AAV carrier shown in the accompanying drawing 25 or any other suitable adenovirus or adeno-associated virus vector.If desired, can be by with having the AAV carrier transduction of a kind of different antibiotics resistance gene (i.e. gene except neomycin resistance gene), make second μ heavy chain allelotrope in the isolating colony suddenlyd change.For isozygotying of selecting to generate knocks out cell, when having corresponding microbiotic, cultivate infected cells.Alternately, can be with the isolating colony of AAV carrier transduction that contains neomycin resistance gene, under the situation that has high density microbiotic (promptly kill hemizygote knock out cell and do not kill to isozygoty knock out the antibiotic concentration of cell), cultivate then.

Embodiment 8: programme the again evidence of defective of the nucleus among traditional nuclear transplantation in cattle embryo

Traditional nuclear transfer technology is produced the newborn infant of the work of low ratio usually.As mentioned below, this poor efficiency may because, to small part be because, the ovocyte of reconstruct can not programme donorcells or donorcells nuclear are with the gene transcription that promotes that ovocyte is grown required gene transcription and suppressed to be unfavorable for to grow

again.Embodiment

11 and 12 has described improved cloning process, and this method can be used to produce in the method for the present invention the transgenic ungulates that has sudden change and randomly have xenogenesis Ig gene in prion gene.

Before the ox embryo implants in the growth course, the distribution of nuclear envelope, nuclear matrix and chromatin-matrix interface composition (chromatin-matrix interface component)

For the distribution among definite kernel tunicle (B-type and A/C-type lamin), nuclear matrix (NuMA) and the embryo of chromatin-matrix interface (AKAP95) composition before implantation, produce the ox embryo by (IVF) in vitro fertilization, and check by immunofluorescence analysis.The in vitro fertilization of ox carried out (Collas etc., Mol.Reprod.Devel.34:212-223,1993) according to previous description.In brief, from the ox seminal fluid layering of the freeze-thaw of a bull the superiors in the 45-90%Percoll gradient, with 700 * g centrifugal 30 minutes.Determine the seminal concentration in the precipitation, the dilution seminal fluid makes that the ultimate density of the time of fertilization is 10 ⁶Sperm/ml.During after maturation 22 hours, the washing ovocyte is 3 times in TL HEPES, is placed in the fertilization substratum of 480 μ l.In 50 ovocytes with 10 ⁶Sperm/ml adds 20 μ l sperm suspensions.In containing the fibroblastic 24 hole tissue culture plate of individual layer mouse fetal, cultivate the embryo, in the 0.5ml embryo culture medium, cover the mineral oil (Sigma) of 0.3ml through embryo testing.Place 25-50 embryo in each hole, under 38.5 ℃ at 5% CO ₂Atmosphere surrounding under cultivate.Detect by former caryogenesis, determine that rate of fertilization surpasses 90%.

For these embryos in vitro fertilization are carried out immunofluorescence analysis, from doctor Jean-ClaudeCourvalin, CNRS, Paris, France obtain the antibody of anti-human nuclear fabric layer albumen B.Buy the monoclonal antibody of anti-Lamin A/C from Santa-Cruz Biotechnology, and obtain the antibody of anti-NuMA from the Transduction laboratory.The rabbit polyclonal antibody of the protein affinity purification of the mouse AKAP95 of the Chinese People's Anti-Japanese Military and Political College derives from Upstate Biotechnologies.Ox embryo attachment in vitro fertilization to the glass cover slide of poly-l-lysine bag quilt, is fixed 15 minutes with 3% Paraformaldehyde 96, change processing thoroughly 15 minutes (Collas etc., J.Cell Biol.135:1715-1725,1996) with 0.1% Triton X-100.With PBS/0.01% Tween 20 (PBST) the closed protein matter that contains 2%BSA 15 minutes.First antibody (anti-AKAP95, anti-lamin B, anti-LBR, anti-NuMA and anti-Lamin A/C) and second antibody were all distinguished incubation 30 minutes, in PBST-BSA, dilute and use with 1:100.With the 0.1Ag/ml Hoechst33342 that is combined in the anti-mounting medium that fades DNA is redyed.Sample is placed on the slide glass, with nail varnish sealing cover slide.On Olympus BX60 surface fluorescence microscope, carry out immunofluorescence and observe, take pictures with JVC CCD photographic camera and AnalySIS software.With AldusPhotostyler software processes photo.Quantizing program with AnalySIS quantizes relatively to fluorescent signal.Data are expressed as average relative intensity of fluorescence.

Ox embryo's immunofluorescence analysis shows, detects Type B lamin (accompanying drawing 29A) in nuclear periphery.And do not detect Lamin A/C in protokaryon phase or 8 cell stages.Being expected at these cell stages early, not detect Lamin A/C be (Guilli etc., EMBO J.6:3795-3799,1987) because the mark of noble cells.All detect nuclear matrix structural protein NuMA (accompanying drawing 29A) at all examination phases.But in the ox embryo of protokaryon phase, the NuMA mark is limited to female pronucleus (FPN), and female pronucleus is less that (arrow among the accompanying drawing 29A) in two protokaryons.AKAP95 is characterized (Bomar etc., 2002 first drafts of submitting to) recently in mice embryonic, and is arrived by Chinese People's Anti-Japanese Military and Political College's mouse AKAP95 antibody test of protein affinity purification, and AKAP95 also only limits to female pronucleus (accompanying drawing 29A).But, in the nucleus of all blastomeres of follow-up etap, also observe AKAP95 at intracellular distribution (accompanying drawing 29A).

Carry out western blot analysis by embryo in vitro fertilization, the specificity (accompanying drawing 29B) of checking immunofluorescence label to ox fetal fibroblast of former generation and protokaryon phase.Analyze for this, 10% SDS-PAGE by the 40mA/ gel comes solubilising protein.Under 100V 1 hour, in transfering buffering liquid (25mM TrisHCl, pH8.3,192mM glycine, 20% methyl alcohol and 0.1% SDS) with protein by electrophoretic transfer to nitrocellulose membrane.With Tris buffer saline (TBS; Be 140mM NaC1,2.7mM KC1 and 25mM Tris-HC1, pH8.0) the washing film is 10 minutes, with the TBST that contains 5% milk (TBS that contains 0.05% Tween-20) sealing 1 hour, and with following first antibody incubation 1.5 hours: anti-AKAP95 (1: 250 diluent), anti-lamin B (1: 1000), anti-LBR (1: 500), anti-NuMA (1: 500) and anti-Lamin A/C (1: 500).The washing spot is 2 times in TBST, 10 minutes, uses horseradish peroxidase (HRP) link coupled second antibody incubation 1 hour.Spot washs in TBST 2 times, 10 minutes, and with the enhanced chemoluminescence (ECL Amersham) shows.

Detect all proteins: 68kDa (Type B lamin), 70 and 60kDa (being respectively nuclear lamina protein A and C), about 180kDa (NuMA) and 95kDa (AKAP95) with the apparent molecular weight of expection.In a word, these results show that the ox embryo express cell nuclear structure albumen before implanting can detect with cross-reacting antibody.Especially, among the ox embryo before implantation, fail to detect Lamin A/C immunely.Because Lamin A/C is expressed (accompanying drawing 29B) in somatocyte, they may constitute the molecule marker that the nucleus in the nuclear transfer embryo is programmed again.

Nuclear envelope, NuMa and the AKAP95 kinetics in nuclear transplantation ox embryo

In ox, detect the nuclear envelope in traditional nuclear transplantation method and the kinetics of nuclear matrix structure.Study these structures with the antibody of Lamin A/C and B, NuMA and AKAP95 respectively.In order to determine that these are marked at the kinetics in the nucleus process of reconstruction, with isolating former generation as discussed previously fetal fibroblast as donorcells, the ox embryo of production nuclear transplantation (Kasinathan etc., Biol.Reprod.64:1487-1493,2001).In brief, by using 0.08% pancreatin and 0.02%EDTA (pancreas enzyme-EDTA) trypsinized that is dissolved among the PBS, harvested cell from the ox fetus.Cell inoculation in α-MEM of T75 culture flask (Corning) (Gibco), has been added 10% foetal calf serum (FBS among α-MEM; Hyclone), 0.15g/ml glutamine (Sigma), 0.003% (3-mercaptoethanol (Gibco) and microbiotic-anti-mycotic agent (Gibco).At postvaccinal the 3rd day, use the pancreas enzyme-EDTA harvested cell, and be chilled among α-MEM/DMSO.The cell (Kasinathan etc., Biol.Reprod.64:1487-1493,2001) of separation G1 phase as discussed previously.In brief, before separation 24 hours are with 5.0 * 10 ⁵Cell is taped against in the T75 flask that contains 10ml MEM/FBS.Second day, dull and stereotyped with PBS washing, change substratum and reach 1-2 hour, middling speed is vibrated dull and stereotyped 30-60 second on Vortex.Remove substratum, with 500x g centrifugal 5 minutes, precipitation is resuspended among the 250 μ l MEM/FBS.With the cell doublet of micropipet selection, and be used for nuclear transplantation by the cytoplasmic bridge connection.

The nuclear transplantation in cattle (Kasinathan etc., Biol.Reprod.64:1487-1493,2001) that carries out as discussed previously.After maturation 18-20 hour, with the ovocyte stoning of maturation in vitro.After the donorcells of G1 phase being transferred to ovum week crack, with electricimpulse fused cell 20 microseconds of single 2.4kV/cm (Electrocell Manipulator 200, Genetronics).28-30 after maturation hour (promptly, ovocyte is collected from ovary that the back was placed 28-30 hour and is merged with donorcells maturation medium after at least 2 hours), activate ovocyte and 4 minutes (Cal Biochem) of parthenogenesis contrast, then (100mM NaC1,3mM KC1, the 0.27mM CaCl in the ACM substratum of reconstruct with 10 μ g cycloheximides and 2.5 μ g cytochalasin D (Sigma) with Calcium ionophore (5 μ M) ₂, 25mM NaHCO ₃, 1mM Sodium.alpha.-hydroxypropionate, 0.4mM pyruvate salt, 1mM L-glutaminate, 3mg/ml BSA, 1% BME amino acid and 1%MEM non-essential amino acid) activate 5 hours (Liu etc., Mol.Reprod.Dev.49:298-307,1998).After the activation, washing nuclear transfer embryo or ovocyte 5 times, and under 38.5 ℃ at 5% CO ₂Cultivate altogether with the mouse fetal inoblast in the air.

Activate the embryo of reconstruct with standard method, merged back 3 hours, the embryo that will be in ripe prochromatin enriching stage fixes with Paraformaldehyde 96, and with the antibody of Lamin A/C, lamin B, NuMA and AKAP95 by immunofluorescence analyze (accompanying drawing 30, PCC).

In addition, the group that allows to grow the protokaryon nuclear transfer embryo in (PN) stage (promptly merging back 15 hours ox embryo) is similarly analyzed (accompanying drawing 30, nuclear transplantation-PN).In contrast, to as said activated parthenogenesis ovocyte also check in the protokaryon stage (accompanying drawing 30, Parth.PN).

As expected, donor somatocyte (bovine fetal fibroblast, accompanying drawing 30) is with underlined as the desired distribution and expression of document institute.At the enriching stage of ripe prochromatin, assemble by the remarkable spissated karyomit(e) that develops with Hoechst 33342 dyeing DNA.On spissated karyomit(e) or near spissated karyomit(e) place, (accompanying drawing 30, PCC), the chances are because they are dispersed in the tenuigenin of ovum not detect Lamin A/C and B.Detect the NuMA that some are labeled; This NuMA probably is associated with the spindle pole of keeping condensation.On the contrary, AKAP95 is associated with spissated (PCC) karyomit(e).This result makes us recalling AKAP95 mark (Collas etc., J.Cell Biol.147:1167-1180,1999 in people's the mitotic cell; Steen etc., J.Cell Biol.150:1251-1262,2000).In the protokaryon phase, it is underlined to detect institute.Lamin A/C is present on the former nuclear envelope (accompanying drawing 2, nuclear transplantation-PN).This lacks this mark and forms and contrast with going up at the tunicle (accompanying drawing 29A) of the protokaryon of the tunicle (accompanying drawing 30) of contrast parthenote (parthenote) protokaryon and fertilization.In the nuclear transplantation protokaryon, detect lamin B, in the contrast protokaryon, also detect this mark.In addition, except kernel, NuMA and AKAP95 are dotted with in nuclear inside.N uMA mark in the nuclear transplantation protokaryon was compared all the time according to bright (comparing nuclear transplantation PN and Parth.PN, accompanying drawing 30) in the parthenogenesis protokaryon.Jointly, these observationss show that the protokaryon of nuclear transfer embryo is ressembled somatic cell nuclear mark nuclear lamina protein A and C, demonstrate intensive NuMA dyeing.

The difference grappling of AKAP95 in the protokaryon of parthenogenesis embryo and nuclear transfer embryo

A-kinases anchorin AKAP95 is the spissated nucleoprotein of hint mitotic chromosome.As influencing the again another kind of molecule marker of programming of somatic cell nuclear after nuclear transplantation, the grappling component is characterized among the ox embryos in the nuclear transplantation protokaryon stage that forms from fetal fibroblast in the nuclear of AKAP95.AKAP95 also has been examined at parthenogenesis embryo's protokaryon and the grappling in the donor somatic cell nuclear.

Come original position to check grappling in the born of the same parents of AKAP95 in the protokaryon embryo by at room temperature extracting the embryo with 0.1% Triton X-100,1mg/ml DNA enzyme I and 100mM or 300mM NaCl.As noted above, male pronucleus does not have any AKAP95.On the contrary, in the nuclear transfer embryo protokaryon and donorcells nuclear of ox, a large amount of AKAP95 and DNA produce resistance (accompanying drawing 31) to DNA enzyme I and 300mM NaCl.In parthenote or nuclear transplantation protokaryon, do not extract Type B lamin (accompanying drawing 31) by DNA enzyme I and 300mM NaCl, show that variation that AKAP95 and DNA distribute is not that overall change owing to the nucleus structure causes.These data show, as in somatic cell nuclear, aspect ox, compare with gynecogenic protokaryon, and AKAP95 more closely is anchored in the nucleus inner structure of nuclear transplantation protokaryon.Whether whether this association retrain dna structure or produce from the genome structure that changes, and still awaits determining.Because the DNA of DNA enzyme I resistance transcribes by silence, the AKAP95 grappling not exclusively reinvents the expression that may slacken important function of gene on growing after the nuclear transplantation.

The transcribing property mistake of Lamin A/C in nuclear transplantation ox embryo regulated

A remarkable result is that Lamin A/C is ressembled in the periphery of nuclear transplantation in cattle embryo protokaryon, and this somatocyte specific marker is not present in protokaryon in vitro fertilization and the gynecogenic protokaryon.Therefore, whether the inventor has studied ressembling of Lamin A/C is because (i) at the somatocyte lamin of ripe prochromatin enriching stage depolymerization target (re-targeting) (accompanying drawing 30) again, (ii) from the translation of the set of maternal Lamin A/C mRNA and assembling or (iii) somatic cell nuclear lamina protein A (LMNA) gene transcribing again in the nuclear transplantation protokaryon.

In order to distinguish these possibilities, by producing the nuclear transplantation in cattle embryo as " tradition " described here nuclear transplantation method, activate the embryo of reconstruct after the nuclear transplantation with inhibitor of protein cycloheximide (CHX), perhaps transcribe again when having rna plymerase ii (PolII) inhibitor radiating streptozotocin D (ActD), activating after the nuclear transplantation suppressing.In order in cycloheximide, to cultivate the nuclear transplantation in cattle embryo, except cultivation ovocyte in cycloheximide (CHX) 14 hours, after nuclear transplantation, activate ovocyte as mentioned above.Activate back 14 hours, washing ovocyte 5 times, and in the ACM substratum that contains 15 μ g/ml Hoechst 33342 (Sigma), placed 1 hour.After the cultivation, by the former caryogenesis of surface fluorescence microscopic examination.Fixing protokaryon embryo in being dissolved in 3% Paraformaldehyde 96 of PBS washs and is placed on the slide glass then.In order in radiating streptozotocin D, to cultivate the nuclear transplantation in cattle ovocyte,, after nuclear transplantation, cultivate ovocyte as mentioned above except in culturing step, in cycloheximide, adding the 5 μ g/ml radiating streptozotocin Ds (ActD).After 5 hours, washing ovum 5 times, and be placed in the ACM substratum that contains 5 μ g/ml radiating streptozotocin Ds.Activate back 14 hours, washing ovocyte 5 times, and in the ACM substratum that contains 15 μ g/ml Hoechst 33342 (Sigma), placed 1 hour.After the cultivation, by the former caryogenesis of surface fluorescence microscopic examination.Fixing protokaryon embryo in being dissolved in 3% Paraformaldehyde 96 of PBS washs and is placed on the slide glass then.

Lamin B is not subjected to protein or the synthetic influence that suppresses of RNA in the assembling of nuclear transplantation protokaryon periphery.This result shows that lamin B obtains from the somatocyte set of previous depolymerization and/or a large amount of lamin B assemblings from the ovocyte kytoplasm.In the inspection of nuclear transplantation protokaryon, measure Lamin A/C (accompanying drawing 30), but in nucleus, lack Lamin A/C with the cycloheximide activating and regenerating.This result shows, the Lamin A/C assembling needs synthetic protein again, and these lamins are transplanted by donor nuclei or the decomposer cell bank of cytogamy from bring ovocyte in guiding again.When activating the embryo when having radiating streptozotocin D, Lamin A/C is not ressembled.These results show, Lamin A/C is ressembled in the nuclear transplantation protokaryon owing to the LMNA gene is transcribed again in the reconstruct protokaryon and produced.In the protokaryon of nuclear transplantation, detect NuMA, and in protokaryon, do not ressemble NuMA, but in the protokaryon of the nuclear transfer embryo that cycloheximide D handles, faintly detect NuMA with cycloheximide activated nuclear transfer embryo.These results show that effectively the assembling of NuMA in the nuclear transplantation protokaryon need take place to translate again, translates at least in part from the set of maternal NuMA mRNA.Compare with the untreated nuclear transfer embryo of contrast, anti-NuMA mark in the nuclear transfer embryo of actinomycin D treatment weak (b ' and the b ' ' ' of accompanying drawing 32 relatively), the result of this unanimity shows that the part NuMA assembling in the nuclear transplantation protokaryon is owing to transcribing again of protokaryon phase NuMA gene produces.

In a word, these results show that after nuclear transplantation, nucleus is reinvented the LMNA gene is closed.Similarly, in the nuclear transfer embryo of protokaryon phase, the NuMA gene obviously still has activity.May be in the chromatin concentration process before ripe, temporary transient inactivation takes place in these genes, and this can infer from the spissated characteristic of chromatinic height and draws (accompanying drawing 30).These results illustrate that clearly in the nuclear transfer embryo of producing, nucleus is programmed not exclusively again under condition described herein.To the discussion of AKAP95, the inventor thinks, continues to exist Lamin A/C can influence genetic expression in the nuclear transplantation protokaryon as previously, for example grows the expression of going up important function of gene.The nuclear lamina protein A of previous report and the interaction of C and chromatin protein and DNA, and this hypothesis of the same support of the relation of these lamins and transcription factor.

Embodiment 9: the defective of programming again of the Exemplary core in traditional nuclear transplantation in cattle embryo

Exemplary difference between natural embryo and the traditional core transplanting embryo also is described hereinafter.These difference comprise the difference that protokaryon assembling, protokaryon NuMA and the conjugated protein concentrated rising of TATA of the A type lamin that noble cells is special and nuclear matrix-karyomit(e) Interface composition AKAP95 and DNA raise to the susceptibility of extracting with washing agent, DNA enzyme and salt.For these research, set up bovine fetal fibroblast system (Kasinathan etc., Nat.Biotechnol.19:1176-1178,2001 and Kasinathan etc., Biol.Reprod.64:1487-1493,2001) as previously described like that.With previously described screening out (shake-off) method, from culture, separate the inoblast doublet (Kasinathan etc., Nat.Biotechnol.19:1176-1178,2001) of G1 phase.As described previously, the ovocyte with maturation in vitro carries out (Collas etc., Mol.Reprod.Dev.34:224-231,1993) in vitro fertilization.For nuclear transplantation (NT) and activation of oocytes, as previous report, in maturation back 20 hours, carry out nuclear transplantation (Kasinathan etc., Nat.Biotechnol.19:1176-1178,2001 and Kasinathan etc. with the donorcells of G1 phase, Biol.Reprod.64:1487-1493,2001).When 28-30hpm (T=0), activate the embryo 4 minutes of reconstruct with 5 μ M Calcium ionophores, then with 10 μ g/ml CHX and the activation of 2.5 μ g/ml cytochalasin D 5 hours.The washing embryo cultivates altogether with the mouse fetal inoblast (Kasinathan etc., Biol.Reprod.64:1487-1493,2001).When in CHX, cultivating the embryo of reconstruct, activate ovocyte as mentioned above, and cultivate 9 hours (in CHX, 14 hours altogether) again with 2.5 μ g/ml CHX.Thoroughly washing embryo, and as discussed previously cultivation the (Kasinathan etc., Biol.Reprod.64:1487-1493,2001).When the embryo of reconstruct is exposed to ActD,, activate ovocyte as mentioned above except in 5 hours CHX culturing step, adding the 5 μ g/ml ActD.

For immunoassay, cell, ovocyte, embryo, nucleus (embodiment 14) and chromatin (embodiment 14) are loaded on the cover glass of poly-l-lysine bag quilt, fix 15 minutes with 3% Paraformaldehyde 96, and with 0.1% saturatingization of Triton X-100 15 minutes.With PBS/2% BSA/0.01% Tween 20 closed protein matter.First antibody and second antibody (dilution in 1: 100) were cultivated respectively 30 minutes.Especially, use the rabbit polyclonal antibody (Chaudhary etc., J.Cel Biol.122:295-306,1993) of anti-human nuclear fabric layer albumen B.Also use available from the anti-lamin B of the goat of Santa-Cruz Biotechnology polyclonal antibody, anti-Lamin A/C monoclonal antibody and anti-TBP antibody.The monoclonal antibody of anti-NuMA derives from Transduction Laboratories, and the monoclonal antibody of the mouse AKAP95 of Chinese People's Anti-Japanese Military and Political College protein affinity purification is available from Upstate Biotechnologies.DNA redyes with 0.1 μ g/ml Hoechst 33342.Take pictures with JVC CCD photographic camera, and quantize immune fluorescence intensity with AnalySIS software.Data are expressed as with respect to the average ± SD fluorescence intensity that contrasts, repeat at least 3 times.For in-situ extraction, before immunofluorescence analysis, the embryo and the cell that are placed on the slide glass were cultivated 15 minutes with 0.1% Triton X-100, the 1mg/ml DNA enzyme I and the 300mM NaCl that are dissolved among the Tris-HCl (pH7.2).For immunoblotting, 100 embryos are dissolved in the 20 μ lSDS sample buffer, use the 10%SDS-PAGE solubilising protein, and analyze: anti-lamin B, 1:1,000 with following antibody; Anti-Lamin A/C, 1:250; Anti-NuMA, 1:500; Anti-AKAP95,1:250.

Based on above-mentioned immunoassay, the distribution in being generally used for the bovine fetal fibroblast of NT characterizes (accompanying drawing 40) to A type/C type and Type B lamin, NuMA and AKAP95.Among the ox embryo before the implantation of external preparation, just in the nucleus periphery, detect lamin B (accompanying drawing 40) as far back as protokaryon (PN) stage.As expected, in the cell of differentiation, lack Lamin A/C.NuMA and AKAP95 only limit in the female pronucleus in protokaryon stage, but in follow-up phase, are dispersed in the whole nucleus.In immunoblotting, confirmed the specificity (accompanying drawing 40) of immunofluorescence data.

The ox inoblast is being transplanted in the nucleus profile remodeling process relevant by electroporation with enucleation oocyte, detect kinetics Biol.Reprod.64:1487-1493 such as (, 2001) Kasinathan of Lamin A/C and B, NuMA and AKAP95.In 3 hours that merge, ripe preceding karyomit(e) takes place and concentrates (PCC) (accompanying drawing 41A) in donor nuclei.Nucleus lamin and NuMA redistribute in the ovocyte kytoplasm, and no longer are present in (accompanying drawing 41A) in the PCC karyomit(e).AKAP95 is relevant with PCC karyomit(e), and this is the characteristic relevant with mitotic cell.After activation of oocytes is handled beginning 14 hours, the NT embryo demonstrates the protokaryon (accompanying drawing 41A, NT PN) of growing fully.But opposite with parthenogenesis embryo or fertilization embryo's protokaryon, in fact all NT protokaryons have intensive Lamin A/C and NuMA immunoreactivity, and this is somatic two features of donor (accompanying drawing 41A).

When having 10 μ g/ml inhibitor of protein cycloheximide (CHX) or 5 μ g/mlRNA polysaccharase (Pol) II inhibitor radiating streptozotocin Ds (ActD) (the two all forms compatible with protokaryon), the assembling (accompanying drawing 41B and 41C) of acceptor activation of oocytes protokaryon Lamin A/C.This result shows that the proteic assembling of these somatocyte nuclear laminas is produced by somatic cell nuclear lamina protein A (LMNA) gene transcription.Lamin B assembling is not subjected to the influence of CHX or ActD, shows this lamin B assembling guiding (accompanying drawing 41B and 41C) again from the maternal storehouse of the somatocyte storehouse of decomposing and/or Type B lamin.

In fact, after exposing, CHX do not detect NuMA; But after handling, in the NT protokaryon, detect 40%NuMA immunoreactivity (accompanying drawing 41B and 41C) with ActD.This result shows that the NuMA assembling is owing to maternal mRNA translation causes with transcribing again.Because Lamin A/C and NuMA are transcribed in the NT protokaryon abnormally, these protein can be used as mark and come programme the again ability of donor genetic material of definite kernel implantation method.

As discussing among the embodiment 8; AKAP95 is a kind of structural multivalence albumen (Collas etc. at nuclear matrix-chromatin interface; J.Cell Biol.147:1167-1179; 1999); enrichment in super acetylizad chromatin, it can be used as a kind of mark that the donor genetic material is programmed again in intracellular grappling character.AKAP95 is unique mark that is studied, and is detected on the karyomit(e) of donor somatic cell nuclear He in the NT protokaryon, has and parthenote (accompanying drawing 41A) and the similar mark intensity of PN embryo (accompanying drawing 40B).By synthesizing of arrestin matter or RNA, kept the relation of AKAP95 and NT protokaryon.Therefore, the major portion of PN AKAP95 is the somatocyte source.Detect the AKAP95 grappling by extracting NT embryo, parthenote and donor inoblast with 0.1% Triton X-100,1mg/ml DNA enzyme I and 300mM NaCl.In parthenote, about 90% AKAP95 and DNA are extracted; But the AKAP95 of about 35% in the NT protokaryon is not extracted (accompanying drawing 41D).AKAP95 and DNA are to the susceptibility of DNA enzyme I and NaCl and the susceptibility similar (accompanying drawing 41D) of inoblast nuclear.Under these conditions, do not extract lamin B, the difference that shows AKAP95 and DNA extraction performance is not to be produced by the total variation in the nuclear structure.These results mean, the NT protokaryon be characterized as tight grappling by AKAP95, and limited DNA is to the proximity of DNA enzyme I.Therefore as if, the protokaryon of producing by somatocyte NT shows as textural anomaly, this be owing to the donorcells nuclear morphology not exclusively reinvent and/or the wrong transcriptional control of somatic cell gene causes.

Embodiment 10: the exemplary cells in traditional nuclear transplantation in cattle embryo is examined the defective of programming again.

Described in embodiment 9, the expression pattern in the pre-implantation embryos of expression pattern among nuclear transplantation (NT) embryo and produced in vitro (IVP) is compared.In order to carry out this comparison, carried out in vitro fertilizationly, and carry out embryo culture (Collas etc., Mol.Reprod.Dev.34:224-231,1993 and Kasinathan etc., Biol.Reprod.64:1487-1493,2001) as described previously.By being fused to, the donor bovine fetal fibroblast carries out NT (Kasinathan etc., Nat.Biotechnol.19:1176-1178,2001 and Kasinathan etc., Biol.Reprod.64:1487-1493,2001) in the non-nucleus egg mother cell.After maturation (hpm) 28-30 hour,, then activate 5 hours, and wash with 10 μ g/mlCHX and 2.5 μ g/ml cytochalasin D with Calcium ionophore activated receptor ovocyte 4 minutes.Embryo and mouse fetal inoblast are cultivated (Kasinathan etc., Biol.Reprod.64:1487-1493,2001) altogether.Handle for CHX, activate ovocyte as mentioned above, before cultivating, cultivated the embryo again 9 hours with 2.5 μ g/ml CHX.Handle for ActD, as mentioned above ovocyte is activated, except adding 5 μ g/ml ActD in 5 hours CHX culturing step, and before cultivating, the embryo was placed 9 hours in 5 μ g/ml ActD again.External embryo culture is arrived the blastaea stage, in every acceptor dam, transplant two pieces of embryos.Monitor gestation by ultrasound investigation, and carry out the C tomoscan.Be born back 24 hours in, by the animal doctor to calf mark (score).

In order to estimate the protein level among NT and the IVP embryo, use available from the anti-lamin B polyclonal antibody of Santa-CruzBiotechnology, anti-Lamin A/C monoclonal antibody and anti-TBP polyclone or monoclonal antibody.The antibody of anti-AKAP95 is from UpstateBiotechnologies.Carry out immunofluorescence analysis (Bomar etc., J.Cell Sci.115:2931-2940,2002) as described.In brief, cell and embryo are placed on the cover glass of poly-l-lysine bag quilt, fix 15 minutes with 3% Paraformaldehyde 96, with 0.1% saturatingization of Triton X-100 15 minutes, closed protein matter in PBS/2% BSA/0.01% Tween 20.Sample was cultivated 30 minutes with first antibody and second antibody (1:100 dilution) respectively.Redye DNA with 0.1 μ g/mlHoechst 33342.Take pictures with JVC CCD photographic camera, and quantize immune fluorescence intensity with AnalySIS software.If desired, before carrying out immunofluorescence analysis, on cover glass, extracted sample 15 minutes with the mixture that is dissolved in 1% Triton X-100 among the Tris-HCl (pH7.2), 1mg/ml DNA enzyme I and 300mMNaCl.For immunoblotting, with 10%SDS-PAGE solubilising protein sample (30 μ g), trace and is detected with specific antibodies to nitrocellulose membrane.

The kinetics of donor nuclei in nuclear transfer embryo

In order to study the kinetics of somatic cell nuclear in nuclear transplantation in cattle (NT), check two constituents of nuclear envelope, by Type B lamin of wide expression (being called lamin B) and the specific A type of noble cells lamin (Lamin A/C) (Gruenbaum etc., J.Struct.Biol.129:313-323,2000).Lamin anchors to nuclear membrane on the chromatin, it is generally acknowledged that it promotes the nucleus expansion after the reconstruct of external (former) nuclear.It is crucial that lamin also is proved to be for cell survival, can cause necrocytosis because can not assemble the Type B lamin.As the mark of the mechanism of transcribing, the kinetics of the conjugated protein TBP of TATA-is analyzed, TBP comes down to all gene transcription factors.Week distribution of the nuclear of lamin B and TBP and the common location (colocalization) of DNA in the pre-implantation embryos of bovine fetal fibroblast and produced in vitro (IVP) are consistent (Holy etc. with observations in other species, Dev.Biol.168:464-478,1995; Houliston etc., Development 102:271-278,1988; With Worrad etc., Development 120:2347-2357,1994).(accompanying drawing 46A).As the mark of noble cells, before implantation, do not detect Lamin A/C (accompanying drawing 46A) in the growth course.Verified the specificity (accompanying drawing 46B) of immunofluorescence label by immunoblotting.

With the inoblast nuclear transplantation behind non-nucleus egg mother cell, Lamin A/C and B be spissated karyomit(e) (PCC before ripe; Merged back 3 hours) on dissociate, and TBP still combine with karyomit(e) (accompanying drawing 46C, PCC).The acceptor Activation of Oocyte is being begun back 14 hours, all NT embryos contain protokaryon that composition with all lamin B marks of nuclear grows and with DNA altogether localized TBP (accompanying drawing 1C, NT-PN).But opposite with the IVP embryo, the NT embryo of 95-99% just expresses Lamin A/C (accompanying drawing 46C) as far back as the protokaryon stage, and at whole early development stage continuous expression (vide infra) all.The fluorescence intensity by measuring the second antibody in the nucleus detected and the ratio (monoploid is than diploid) of the DNA fluorescence intensity (Hoechst 33342) of representation DNA concentration, in quantification NT embryo and IVP embryo's the protokaryon by the relative content of immune labeled lamin B, Lamin A/C and TBP.Though in NT embryo and IVP embryo's protokaryon, the relative content of lamin B is that similarly Lamin A/C in the NT protokaryon and the relative content of TBP are than IVP embryo's the male pronucleus (MPN) or the height (accompanying drawing 46D) of female pronucleus (FPN).

TBP in NT embryo's protokaryon, DNA and A type lamin

The TBP of the high level in the NT protokaryon is with relevant to the big resistance of carrying out in-situ extraction with the combination of washing agent (1% Triton X-100), nuclease (1mg/ml DNA enzyme I) and salt (0.3M NaCl).Immunofluorescence label intensity with respect to the contrast that is not extracted, immunofluorescence label intensity among the embryo who quantizes to be extracted, the result shows, in IVP embryo's male pronucleus (MPN) or female pronucleus (FPN), 35% the TBP of still having an appointment is not extracted (accompanying drawing 47A).But as in the inoblast nuclear, the TBP in the NT protokaryon has very strong resistance (accompanying drawing 47A) to extraction.Similarly, compare with IVP embryo's protokaryon, the DNA of NT protokaryon has raise 4.5 times to the resistance of the extraction under these conditions, and (accompanying drawing 47A DNA), has pointed out chromatin Structure more closely.

In order to determine the source of lamin B, Lamin A/C and TBP in NT embryo's protokaryon, as described herein, there is RNA polymerase (Pol) II inhibitor radiating streptozotocin D (ActD; 5 μ g/ml) time or use inhibitor of protein cycloheximide (CHX, 10 μ g/ml) that the acceptor ovocyte is activated (Knott etc., Biol.Reprod.66:1095-1103,2002).The assembling of Lamin A/C, lamin B and TBP is checked by the protokaryon embryo of photodensitometry immunofluorescence label.These two kinds of inhibitor all stop the assembling (accompanying drawing 47B and 48C) of Lamin A/C, show that these somatocyte lamin assemblings in the embryo are that the nuclear lamina protein A gene is transcribed generation in the protokaryon stage.Lamin B assembling is not subjected to the influence (accompanying drawing 47B and 47C) that CHX or ActD handle, and shows that lamin B is dissolved in that somatocyte lamin assembling the ovocyte kytoplasm obtains behind NT and/or assembles from the female parent set of lamin to obtain.In not processed embryo or after suppressing RNA or protein synthesis, also detect the TBP (accompanying drawing 47B and 47C) of similar content.Because TBP is relevant with spissated karyomit(e) in the PCC process, and owing to mid-term II ovocyte kytoplasm lack detectable TBP fully, in the NT process, the TBP that somatocyte is originated may be still relevant with the donor gene seat.Therefore, ultrawhite except expressing A type nuclear fabric layer protein, NT embryo protokaryon has the TBP and the interior grappling enhanced TBP of cell of high level.

Embodiment 11: use the donor chromatin of programming again to come cloning mammal

For the problem of programming not exclusively again that exists in the traditional core transplanting embryo that overcomes illustration in embodiment 8-10, the method that exploitation makes new advances, with the donor chromatin of before nuclear transplantation, programming again more effectively (PCT/USO1/50406, application on December 21 calendar year 2001).These methods are included in the nucleus of cultivating in the substratum of programming again (for example cell extract) from donorcells (nucleus of the heteroantibody of for example encoding), cause the nuclear envelope dissolving, and possible chromatin concentrate.This nuclear envelope disintegration and chromatin concentrate and make the transcriptional regulation protein that is combined on the karyomit(e) be released, and these transcriptional regulation proteins also will promote the gene transcription that ovocyte, embryo or fetation are not expected.In addition, the modulin from the substratum of programming again will be attached on the chromatin and the required gene transcription of promotion growth.

In order to produce the ungulate that on prion gene, has sudden change and selectively have xenogenesis Ig gene, can again the programming before, during or afterwards, by one or more allelotrope of sudden change Protein virus with selectively by inserting the nucleic acid of one or more coding heteroantibody, modify donorcells nuclear or chromatin.If desired, be used to second from the cell of cloning fetus or clone offspring and take turns nuclear transplantation, come the outer clone's of delivery capacity offspring.Can also be freezing from the cell of cloning fetus or clone offspring first, form clone as the donorcells source of the outer ungulate of cloning of delivery capacity.

The donorcells nuclear that batch process is used to clone

From cultivate synchronization or the cell colony that is not synchronized in can isolate and reach millions of nucleus.Cell colony synchronization or through the chemical treatment synchronization naturally.Preferably, at least 40 in the colony, 60,80,90 or 100% cell are stuck in G ₀Phase or G ₁Phase.In order to realize this goal, cell cultures in low blood serum medium for example, is for example reached 1,2,3 or more days in 5%, 2% or 0% the serum, improve and be in G ₀The ratio of the cell of phase.For with cell synchronization at G ₁Phase, cell can be grown into the attached cell form and converge, as discussed previously then, at 0.5-1 μ g/ml Luo Keda azoles (SigmaChemicals, St.Louis, MO) cultivate 17-20 hour (referring to, J.Cell Biol.147:1167-1180 such as Collas for example, 1999 and reference wherein) in.By patting the flask that contains the cell that adheres to repeatedly with a hand, the flask that vibrates tempestuously makes mitotic cell and G ₁The doublet of phase separates.G ₁The doublet of phase is the atomization paired extended cell in latter stage, and they still are connected together by a superfine bridging.According to this distinctive doublet structure, can be with isolating G ₁The phase doublet is separated from substratum.After the separation, the doublet of G1 phase can still be preserved connection or may be divided into two cells that separate.

Use standard method, in the physiological saline (PBS) of phosphoric acid buffer, gather in the crops cell synchronized or that be not synchronized, take a plurality of washing steps that cell is transferred to hypotonic buffer liquid (10mM HEPES, pH7.5,2mM MgCl from its original substratum ₂, 25mM KCI, 1mM DTT, 10 μ M press down enzyme peptide, bright enzyme skin, 10 μ M Pepstatin A, 10 μ M Trypsin inhibitor SBTIs and the 100 μ M PMSF of pressing down of 10 μ M) in.For example, can under 4 ℃, come sedimentation cell in centrifugal 10 minutes with 50ml PBS washing with 500x g.Decant the PBS supernatant liquor, precipitation is resuspended among the 50ml PBS, carry out centrifugal as mentioned above.After centrifugal, sedimentary cell is resuspended in the ice-cold hypotonic buffer liquid of 20-50 volume under 4 ℃ centrifugal 10 minutes with 500x g.Decant the P supernatant liquor, add the hypotonic buffer liquid of about 20 volumes in the cell precipitation.Carefully cell is resuspended in this damping fluid,, cell is progressively expanded on ice cultivating at least 1 hour.

For separating nucleus from cell, use the standard method dissolved cell.For example, the cell suspending liquid of 2-5ml is transferred in the glass refiner, the pestle that is fit to size carries out DounceShi homogenate with 10-20 time initial bump.Alternately, come the homogenate cell suspending liquid with mobile agitator (for example Ultraturrax).If desired, monitor cell lysates with phase microscope down at 40 times.In this homogenate process, nucleus should be kept perfectly, and for example vesica, organoid and protein discharge from nucleus for major part or preferably all original kytoplasm compositions that adheres to.If desired, 1-20 μ g/ml cytoskeleton inhibitor cytochalasin B or cytochalasin D are added in the aforesaid hypotonic buffer liquid, to make things convenient for this processing.Homogenate is long enough, discharges the kytoplasm composition with dissolved cell and from nucleus.For some cell type, need nearly 100,150 or more times bump.Then lysate is transferred in the 15ml conical tube that is placed on ice, remaining turgid cell suspension is repeated cytolysis processing.Sucrose mother liquor with the 2M of hypotonic buffer liquid preparation is added in the cell lysates to (for example, the 2M mother liquor of 1/8 volume being added in the lysate), obtain the ultimate density of 250mM sucrose.Mix this solution by putting upside down, by under 4 ℃ in pendulum roller to come precipitate nucleus in the centrifugal 10-40 of 400x g minute.Abandoning supernatant then is resuspended in sedimentary nucleus nucleus damping fluid (10mM HEPES, pH7.5, the 2mM MgCl of 10-20 volume ₂, 250mM sucrose, 25mM KCl, 1mM DTT, 10 μ M press down enzyme peptide, 10 μ M leupeptins, 10 μ M Pepstatin A, 10 μ M Trypsin inhibitor SBTIs and 100 μ M PMSF) in.As mentioned above, precipitate nucleus, and be resuspended in the nucleus damping fluid of 1-2 volume.Fresh separating nucleus can be used for following external programming and nuclear transplantation more immediately, perhaps preserves standby.For preservation, nucleus is diluted in the nucleus damping fluid, to about 10 ⁶The concentration of/ml.Add glycerine (100% glycerine of 2.4 volumes), by pressure-vaccum thorough mixing gently.Suspension is distributed on ice the 1.5ml test tube with the volume of 100-500 μ l, in methyl alcohol-the dry ice bath, carries out freezingly immediately, be kept under-80 ℃.Before the use, the nuclear aliquots containig of thawing on ice or under the room temperature.Add the ice-cold nucleus damping fluid of 1 volume, in pendulum roller with 1, centrifugal this solution of 000xg 15 minutes.Sedimentary nucleus is resuspended in the 100-500 μ l nucleus damping fluid, carries out centrifugal as mentioned above.Sedimentary nucleus is resuspended in the nucleus damping fluid of minimum volume then, preserves standby on ice.

Be used for programme again the mitotic division extract of donor genetic material or the preparation of substratum

Preparation for the mitotic division extract, by in 0.5-1 μ g/ml Luo Keda azoles, cultivate 17-20 hour (referring to, Collas etc. for example, J.Cell Biol.147:1167-1180,1999 and reference wherein) with (for example inoblast) synchronization of somatocyte system at m period, as mentioned above, separate mitotic cell by thermal agitation.Discard isolating G ₁The doublet of phase, perhaps this doublet can keep with the mitotic cell of the major portion that constitutes isolated cell (usually at least 80%) together.Under 4 ℃, in the 10ml conical tube, reach 10 minutes with the isolated cell of the centrifugal results of 500xg.Compile a plurality of cell precipitations, be resuspended among the ice-cold PBS of cumulative volume 50ml, and under 4 ℃ with 500xg centrifugal 10 minutes.Repeat this PBS washing step.Cell precipitation is resuspended in ice-cold cytolysis damping fluid (20mM HEPES, pH8.2, the 5mM MgCl of about 20 volumes ₂10mM EDTA, 1mM DTT, 10 μ M press down the enzyme peptide, 10 μ M leupeptins, 10 μ M Pepstatin A, 10 μ M Trypsin inhibitor SBTIs and 100 μ M PMSF and 20 μ g/ml cytochalasin Bs selectively) in, by under 4 ℃, coming sedimentation cell in centrifugal 10 minutes with 800xg.Abandoning supernatant is resuspended in cell precipitation in the cytolysis damping fluid that is no more than 1 volume carefully.Cell was cultivated on ice 1 hour, made cell expansion.Carry out ultrasonication or carry out Dounce homogenate with glass mortar and pestle and come dissolved cell with most advanced and sophisticated ultrasonoscope.Dissolved cell is dissolved up at least 90% cell and nucleus, and this can evaluate by phase microscope.Being used to dissolve at least 90% cell and nuclear ultrasonic wave time will change according to the type of the cell that is used to prepare extract.

Cell lysates is placed in the centrifuge tube of 1.5ml, with desk centrifuge under 4 ℃ with 10,000 to 15, centrifugal 15 minutes of 000xg.From whizzer, take out test tube, be put on ice immediately.Collect supernatant liquor carefully with 200 μ l transfer pipet tips, will pool together, be placed on ice from the supernatant liquor of a plurality of pipes.This supernatant liquor is " mitotic cell matter " or " MS15 " extract.This cell extract can be distributed in the test tube that is placed on ice, every pipe 50 μ l or 10 μ l depend on that being used to produce chromatinic is ordinary method or micromethod.Rapid freezing this extract in liquid nitrogen immediately, and preserve up to use down at-80 ℃.Alternately, cell extract is added on ice the ultracentrifugation pipe and (for example, is suitable for the SW55Ti rotor; Beckman).If desired, in test tube, cover mineral oil up to the top.With 200, the centrifugal extract of 000xg 3 hours precipitates the membrane vesicle that is included in the MS15 extract under 4 ℃.In the centrifugal later stage, discard mineral oil.Collect supernatant liquor carefully, if desired, pool together, be added in the cold 1.5ml test tube on ice.This supernatant liquor is referred to as " MS200 " or " mitotic division kytoplasm " extract.The extract mode that is used for MS15 as described is with this extract five equilibrium and freezing.

If desired, can be with other nucleus factor extract-enriched.For example, can be from the cell of the cell type in regeneration extract source or from the cell purification nucleus of any other cell type, and dissolve by ultrasonic wave as mentioned above.Under stirring, in containing NaCl that concentration is 0.15-800mM or KCl nucleus damping fluid, cultivate and extracted the nucleus factor in 10-60 minute.Centrifugal lysate precipitates insoluble composition.Dialysis contains the interested supernatant liquor that is extracted the factor and removes NaCl or KC1.Nucleus extract five equilibrium and freezing preservation with dialysis.Before adding is used for the regenerated nucleus, this nucleus extract is added in the above-mentioned complete cell extract with various concentration.

Can also from sexual cell, prepare mitotic division extract, for example ovocyte or male sex-cell.For example, collect nature be stuck in this stage mid-term II ovocyte, washing and carry out born of the same parents as mentioned above and separate, the parent cell extract of laying eggs next life.In order to prepare the male sex cell extract, by the chopping organ and in sucrose or percoll gradient the cell of gathering in the crops is carried out differential centrifugation, from the testis that the slaughterhouse obtains, separate sexual cell.From body (Leydig and Sertoli) cell, separate sexual cell,, in PBS, precipitate by the washing that suspends.Then as mentioned above in ice-cold cytolysis damping fluid washed cell once and dissolve by ultrasonic wave.By under 4 ℃ with 15,000xg made lysate clarification in centrifugal 15 minutes, five equilibrium supernatant liquor (being the sexual cell extract), and being chilled in the liquid nitrogen rapidly.

As substituting of cell extract, can also for example add in the solution in the damping fluid by the factor (for example nucleic acid or protein for example dna methyltransferase, histone desaminase, histone, protamine, lamin, transcription factor, activator, repressor, Chromatin Remodeling albumen, somatomedin, interleukin, cytokine or other hormones) of one or more are natural or reorganization, prepare the substratum of programming again.Preferably, one or more factor pair ovocytes or stem cell are special.

Spissated chromatinic formation

Thaw the on ice aliquots containig of MS15 or MS200 extract or mitotic division substratum.(0.6 μ l) adds in 20 μ l extracts or the substratum with the ATP-generation structure, and mixes by vibration.For the preparation of ATP-generation structure, mix 100mM ATP mother liquor, the 1M phosphocreatine of equal proportion and the 2.5mg/ml sarcosine kinase solution (100x) that in water, prepares, preserve up to use on ice.Add the ATP generation structure in extract, ultimate density is 1mM ATP, 10mM phosphocreatine and 25 μ g/ml sarcosine kinases.

Nucleus suspension is added in extract or the substratum, and concentration reaches 1 μ l nucleus in per 10 μ l extracts or the substratum, by the pressure-vaccum thorough mixing, cultivate in 30,33,35,37 or 39 ℃ water-bath.Pat at regular intervals and play the test tube that contains mixture, be condensed into piece in the test tube bottom to prevent karyomit(e).For example per at regular intervals 15 minutes, the disintegration and chromosomal the concentrating of microscopically monitoring nuclear envelope.When nuclear envelope disintegration and karyomit(e) have begun to concentrate, begin from extract or substratum, to reclaim chromatinic operation.

Alternately, by pre-loadedly carrying out aforesaid operations after to nuclear matrix protein NuMA isolated cells being examined, concentrate or the chromatin of partial concentration (Steen etc., J.Cell Biol.149,531-536,2000) only to form with antibody.This operation makes and can remove the nucleus composition from chromatin by the nuclear membrane of dissolving around donorcells nuclear; But, suppress enrichment step by adding anti-NuMA antibody.The danger that stops karyomit(e) to concentrate to be reduced in karyomit(e) generation disintegration when cultivating karyomit(e) in the mitotic division extract or lose.

For this operation, the nucleus (2,000 nucleus/μ l) 15 minutes of saturatingization purifying in containing 500 μ l nucleus damping fluids of 0.75 μ g/ml lysolecithin at room temperature.By adding 3%BSA that 1ml prepares and cultivated the excessive lysolecithin of dieing out 5 minutes in the nucleus damping fluid on ice.Precipitate nucleus then, and washing is once in the nucleus damping fluid.Nucleus is resuspended in the (dilution in 1: 40 of anti-NuMA antibody of containing of 100 μ l with 2,000 nucleus/μ l; Transduction Laboratories) in the nucleus damping fluid.Cultivated 1 hour on ice, stir gently, with 1M sucrose with 500xg precipitate nucleus 20 minutes.Then nucleus is resuspended in the nucleus damping fluid, and adds in the mitotic division extract or substratum as the said ATP of the containing generation structure of previous chapters and sections.Selectively, anti-NuMA antibody is added in extract or the substratum, concentrate further to prevent karyomit(e).

Form spissated in washing agent or the protein kinase or the karyomit(e) of partial concentration only by being exposed to.The not combined or loosely that washing agent is used for dissolved cell nuclear is attached to the nucleus composition on the karyomit(e), to remove nuclear envelope.For this operation, for example cultivate the nucleus (2,000-10,000 nucleus/μ l) that is purified in the nucleus damping fluid of 0.1%-0.5% Triton X-100 or NP-40 having added washing agent.For the ease of removing nuclear envelope, other salt are NaCl for example, with about 0.1,0.15,0.25,0.5,0.75 or the concentration of 1M be added in the damping fluid.In shaking culture after 3060 minutes gently on ice, by in pendulum roller with 1, came precipitate nucleus in the centrifugal 10-30 of 000xg minute, centrifugation time depends on cumulative volume.Sedimentary nucleus is resuspended in the 0.5-1ml nucleus damping fluid, and precipitates as mentioned above.Repeat this washing step twice, to guarantee removing washing agent and extra salt fully.

Alternately, with the reorganization or natural protein kinase remove nuclear envelope individually or in combination.Preferably, come purifying protein to swash alcohol with standard method or obtain the protein kinase of purified form from commercial source.These sharp alcohol may make nuclear envelope, nuclear matrix or chromatinic composition phosphorylation, with remove nuclear envelope (referring to, for example Collas and Courvalin, Trends CellBiol.10:5-8,2000).Preferred kinases comprises kinases 1 (CDK1), protein kinase C (PKC), protein kinase A (PKA), the map kinase of cyclin dependent, kinases (CamKII) and the CK1 casein kinase or the CK2 casein kinase of dependence calcium/calmodulin.For this method, under the room temperature, in the 1.5ml centrifuge tube, about 20,000 purified nucleus are cultivated in 20 μ l phosphorylation damping fluids.The preferably phosphoric acid damping fluid that is used for CDK1 (Upstate Biotechnology) contains 200mM NaCl, 50mM Tris-HCl (pH7.2-7.6), 10mMMgS04,80mM β-Phosphoric acid glycerol esters, 5mM EGTA, 100M ATP and 1mM DTT.For PKC, a kind of preferred buffer contains 200mM NaCl, 50mM Tris-HCl (pH7.2-7.6), 10mM MgS0 ₄100 μ M CaCl ₂, 40 μ g/ml phosphatidylserines, 20 μ M triglycerides, 100 μ M ATP and 1mM DTT.If use PKC and CDK1 simultaneously, adopt the CDK1 phosphorylation damping fluid that has added 40 μ g/ml phosphatidylserines and 20 μ M triglycerides.The preferably phosphoric acid damping fluid that is used for PKA comprises 200mM NaCl, 10mM MgS0 ₄, 10mM Tris, pH7.0,1mM EDTA and 100 μ M ATP.For map kinase, use and added 10mM CaCl ₂PKA phosphorylation damping fluid with 1mM DTT.For CamKII, can use PKA damping fluid that has added 1mM DTT or the Cam kinases assay kit of buying from Upstate Biote chnology (Venema etc., J.Biol.Chem 272:28187-90,1997).

By adding the ultimate density of protein kinase, begin phosphorylation reaction to 25-100ng.This reaction is at room temperature cultivated and is reached 1 hour.In this process, by the disintegration of microscope monitoring nuclear envelope, for example with 15 minutes interval.After the nuclear envelope disintegration, washed cell nuclear is 3 times as mentioned above, to remove washing agent solution.

Chromatinic recovery

To contain in the 1M sucrose that spissated, partial concentration or unconcentrated chromatinic extract or solution joins equal volume with the preparation of nucleus damping fluid.Under 4 ℃, in pendulum roller,, precipitated chromatin in the centrifugal 10-30 of 000xg minute with 1, centrifugation time depends on the volume of sample.Abandoning supernatant, carefully sedimentary chromatin being resuspended in 0.1-1.0ml nucleus damping fluid or fat by piping and druming merges in the damping fluid (but being dissolved in 150mM NaCl, 10 μ M among the 20mM MES of the 20mM HEPES of about pH7.0 or pH7.5 or about pH6.2 enzyme peptide, 10 μ M leupeptins, 10 μ M Pepstatin A, 10 μ M Trypsin inhibitor SBTIs and 100 μ M PMSF), and with 1, the centrifugal 10-30 of 000xg minute.Abandoning supernatant is resuspended in the nucleus damping fluid with sedimentary chromatin or fat merges in the damping fluid, preserves up to use on ice.Various chromatin are transferred in the drop of HEPES buffer culture medium of 20 μ l, in the micrurgy culture dish, covered these drops with oil.As described below, a karyomit(e) is inserted in each non-nucleus egg mother cell.

Prepare chromatinic micromethod

With MS15 or the MS200 extract of 10-20 μ l or contain ATP generation structure, washing agent and/or salts solution or mitotic division substratum or aforesaid protein kinase solution are placed in the culture dish.Isolating G1 phase cell doublet or the G in the substratum of being in that adds 50 μ l then ₀The drop of the cell of phase, be used for the convenient cytolytic 0.1%Triton of containing X-100 or the HEPES of NP-40 or isolating 50 μ l " born of the same parents separate " drops of bicarbonate buffer, and the ovocyte of 50 μ l injection nutrient solution drop.Each of these drops CO ₂Equilibrated mineral oil is covered with.In culture dish, also add the substratum " washing drop " of 50 μ l, be used to wash dissolved cell or nucleus.

With transfer pipet cell transfer is separated in the drop to born of the same parents.By repeating pressure-vaccum lightly, dissolved cell film in transfer pipet.When cell is dissolved, lysate is discharged in the washing drop lightly, rapidly nucleus is siphoned away to remove washing agent.Selectively, change the nucleus of processing thoroughly, and before being added to mitotic division extract or substratum, cultivate with anti-NuMA antibody.Nucleus is repelled in the drop of MS15, MS200 or substratum, washing agent and/or salts solution or protein kinase solution then.Monitoring nucleus disintegration and karyomit(e) as mentioned above concentrates.If use the mitotic division extract that does not contain anti-NuMA antibody, in case the nuclear envelope disintegration, karyomit(e) just begins to concentrate, and is as described below, separates single complete chromatin with micropipet, and transfer in the acceptor ovocyte of stoning

The stoning of ovocyte

Preferably, the acceptor ovocyte is the ovocyte of II in mid-term.In this stage, ovocyte is activated or is fully activated, to handle the chromatin of importing as treating the fertilization sperm.For the stoning of ovocyte, part in the ovocyte or preferred all DNA are removed or deactivation.The genetic material that the destruction of this DNA in the acceptor ovocyte or removal have stoped ovocyte exerts an influence to the g and D of cloning mammal.A kind of method that is used for destroying the protokaryon of ovocyte is to be exposed to ultraviolet ray (Gurdon, in Methods in CellBiology, Xenopus Laevis:Practical Uses in cell and Molecular Biology, Kay and Peng, editor, Academic Press, California, 36 volumes: 299-309,1991).Alternately, by any standard technique, exenterate ovocyte protokaryon (referring to, for example McGrath and Solter, Science 220:1300-1319,1983).In a kind of possible method, syringe needle is inserted in the ovocyte, nucleus is drawn onto in the inner chamber of syringe needle.From ovocyte, take out syringe needle then.And do not destroy plasma membrane (United States Patent (USP) 4,994,384 and 5,057,420).

Be used for chromatin is inserted the fat fusion of ovocyte

Merge or microinjection or electric integration technology (referring to for example United States Patent (USP) 4,994,384 and 5,945,577) by standard by fat as described below, chromatin is imported in the acceptor ovocyte.Following fat fusion method can also be used to other application, and karyomit(e) is inserted in other recipient cells.

As mentioned above, from mitotic division extract, washing agent and/or salts solution or protein kinase solution, separate chromatin, merge the damping fluid washing with fat then by centrifugal.Chromatin is stored in ice-cold fat to be merged in the damping fluid up to use.Alternately, five equilibrium chromatin, at liquid nitrogen or freezing in methyl alcohol-the dry ice bath, and under freezing being kept at-80 ℃.One or more gene fusion reagent and fat are merged damping fluid to be respectively the mixed of about 5:1-1:10, and preparation fat merges solution.Gene fusion reagent is by still for example being not limited to polyoxyethylene glycol (PEG) and lipophilic compound ,

,

N-[1-(2,3-two oily acyloxy) propyl group]-N, N, N-trimethyl ammonium Methylsulfate; C ₄₃H ₈₃NO ₈S},

2,3-two oily acyloxy-N-[2 (spermine formamido-) ethyl]-N, N-dimethyl-l-third ammonium trifluoroacetate } and

(dioleoyl phosphatidylethanolamine).

Other preferred lipids comprise neutral and monovalent or polyvalent cation lipid, for example contain those lipids of quaternary ammonium group.Other preferred lipids contain cholesterol moiety, for example those lipids that formed by hydroxyl in the cholesterol and the radical reaction in the lipid.Also have other preferred lipids to have saturated or unsaturated fatty acids, it preferably contains 5 to 10,10 to 15,15 to 20 or 20 to 30 carbon atoms, comprises endpoint value.These lipids can be synthetic with the chemical synthesising technology of standard, obtains from natural origin, perhaps buys and obtain (Biophys such as Summers J.71:3199-3206,1996 from commercial source; Nabekura etc., Pharm Res.13:1069-1072,1996; Walter etc., Biophys J.66:366-376,1994; Yang etc., Biosci Rep.13:143-157,1993; Walter and Siegel, Biochemistry.6:32:3271-3281,1993).Other preferred gene fusion compounds are phosphatide, for example from the membrane vesicle fragment (Collas and Poccia, J.CellScience 109:1275-1283,1996) in sea urchin ovum or other sources.Preferably, karyomit(e) is contacted with the membrane vesicle fragment can not make karyomit(e) by complete film bag quilt.

For cation lipid, for example

, can be used for fat with the concentration of 0.1-30 μ g/ml and merge damping fluid.Alternately, for example can use by cation lipid and neutral lipid

The liposome prescription formed of mixture.

Chromatin prepared fresh or freeze-thaw and fat are merged solution mixes, make and wrap by this compound on this chromatin.Under 20-30 ℃ of temperature, cultivated about 10-30 minute.The chromatinic droplet that contains that fat is merged in the solution is placed on through CO ₂Under the mineral oil that balance is crossed.Also preparation contains the drop of the acceptor ovocyte of stoning.With bag by the chromatin of fat syncretization compound select in the micropipet, be inserted into ovum week between ovocyte kytoplasm and the zona pellucida in the crack.Chromatin is placed near oocyte membrane, contacts with ovocyte guaranteeing.Chromatin-ovocyte mixture maintained under 20-30 ℃ the temperature, merge in the microscopically monitoring.In case merge the ovocyte of activation reconstruct as described below.

The Activation of Oocyte of reconstruct, cultivation and transplanting

In order to prevent that polar body from discharging and chromosome elimination, when having cytochalasin B, activate ovocyte, perhaps after activation, add cytochalasin B (PNAS96:14984-14989 such as Wakayama, 1999 immediately; Nature Genetics 24:108-109 such as Wakayama, 2000).Can adopt electrical or non-electrical means to activate the ovocyte of reconstruct.The power technology that is used to activate ovocyte be in the art know (referring to, for example United States Patent (USP) 4,994,384 and 5,057,420).The non-power technology that is used for activating cells can comprise any technology that is used to improve the cytodifferentiation probability that this area is known.The example that being used to of non-electricity activated the means of ovocyte is included in ethanol; The inose triphosphoric acid; Ca ⁺⁺Ionophore and kinases inhibitor; Protein synthesis inhibitor; Phorbol ester; When any composition of thapsigargin or sperm exists, cultivate ovocyte.Other are used for the non-method for electrically of activated and comprise and make ovocyte through cold shock or mechanical stress.Alternately, after the 1-3 after the nuclear transplantation hour, containing Sr ²⁺Substratum in cultivate ovocyte and activated them in about 6 hours, prevent cytoplasmic move and polar body is discharged (PNAS 96:14984-14989 such as Wakayama, 1999 with cytochalasin B; Nature Genetics 24:108-109 such as Wakayama, 2000).According to the mammiferous type of being cloned, preferably activating length can change.For example, for example in the ox, the activation of oocytes stage is usually at about 16-52 hour or preferred in about 28-42 hour scope domestic animal.

After the activation, ovocyte is placed on reaches appropriate time quantum in the substratum, so that the fetal development that generates.In two cell stages or later stage, that embryo transfer is in the replace-conceive recipient female animal, mature to grow.For ox, in being implanted to the parent host before, usually with embryo culture to blastula stage (for example, about 6-8 days).For other cloned animals, the appropriate length of vitro culture is that those skilled in the art know, and perhaps can determine by routine test.

The method that is used for the embryo is implanted to the Mammals uterus also is well known in the art.Preferably, embryo's etap is relevant with the oestrus cycle of host mammal.In case the embryo is implanted in the mammiferous uterus, this embryo can grow mature.Alternately, the embryo can grow in the uterus selected period, and the operation method with standard takes out embryo (or fetus) then, to determine its healthy state and developmental potency.Can be implanted in the uterus environment from the animal of another species from the embryo of species.For example, the ox embryo can grow (Stice and Keefer, Biology of Reproduction 48:715-719,1993) in the uterine tube of sheep.Any species hybridization relation between embryo and the uterus can be used in the method for the present invention.

The fat of nucleus and ovocyte or other recipient cells merges

As mentioned above, prepare fat fusion solution by one or more gene fusion reagent and fat being merged damping fluid with the various mixed of about 5:1-1:10.Nucleus prepared fresh or freeze-thaw and fat are merged solution mixes, make and wrap by this compound on this nucleus.Under 20-30 ℃ of temperature, cultivated about 10-30 minute.The nucleolate droplet that contains that fat is merged in the solution is placed on CO ₂Under the mineral oil that balance is crossed.Also preparation contains the drop of the recipient cell of the preferred stoning of recipient cell.As mentioned above, mechanically remove karyomit(e) or nucleus or destroy genetic material, prepare the recipient cell of stoning by being exposed in the UV light by micrurgy.For being inserted in the ovocyte, with bag by the chromatin of fat syncretization compound select in the micropipet, be inserted into ovum week between ovocyte kytoplasm and the zona pellucida in the crack.In order to be inserted in other recipient cells, preferably coated nucleus is placed near cytolemma, to guarantee and cells contacting.Nucleus-cell complexes maintained under 20-30 ℃ the temperature, merge in the microscopically monitoring.In case merge, activate the ovocyte of reconstruct as mentioned above.

Embodiment 12: the purposes that the cell of using the process of programming again to change processing thoroughly comes cloning mammal

Also can need not from cell separating nucleus or the chromatin cell of programming again.In the method, with cell permeabilization, under the condition that allows generation factor exchange between substratum (for example cell extract) and the cell, in the substratum of programming again interval or m period, cultivate then.If use the interval substratum, then the nucleus in the cell still combines with film; If use the mitotic division substratum, nuclear envelope disintegration and chromatin then may take place concentrate.By after in this substratum, cultivating the nucleus of programming again, preferably seal plasma membrane again, form the complete cell of programming again, it contains the expecting factor from substratum.If desired, as described in embodiment 11, with other this substratum of nucleus factor enrichment.The cell and the acceptor ovocyte of programming are again merged, embryo's implant carrier receptor body Mammals that will generate, to produce cloning mammal from the ovocyte of reconstruct.

In order in prion gene, to produce the ungulate that has sudden change and randomly have xenogenesis Ig gene, before programming again, during or afterwards, by one or more allelotrope of sudden change prion gene with randomly modify donorcells by inserting one or more nucleic acid of encoding heteroantibody.If desired, will be used for second from fetus of cloning or clone offspring's cell and take turns nuclear transplantation, with the outer clone's of delivery capacity offspring.Can also confess one's crime the in the future fetus of time cloning or clone offspring's cell freezing form the clone as the source of the donorcells of the outer clone ungulate of delivery capacity.

The saturatingization processing of cell

The cell of can programming again in this way comprises the cell that is not synchronized or is synchronized at G ₀, G ₁, S, G ₂Or the combination of the cell of M phase or these stages of cell.Change the processing cell thoroughly with standard method, for example change processing thoroughly with digitonin or streptolysin O.In brief, use the standard method harvested cell, and wash with PBS.Change processing thoroughly for digitonin, cell is resuspended in the substratum of the digitonin that contains the about 0.001-0.1% of concentration, cultivated on ice 10 minutes.For changing processing thoroughly with streptolysin O, at room temperature, streptolysin O solution (referring to, for example FASEB such as Maghazachi J.11:765-774,1997 and reference wherein) in about 15,30 or 60 minutes of culturing cell.Through after the various cultivations, by coming washed cell in centrifugal 10 minutes with 400xg.By resuspended in PBS and precipitation, repeated washing step twice.At room temperature cell is remained among the PBS up to use.Preferably, as described below, will join immediately in interval or the m period substratum through the cell of saturatingization and programme again.

Programme the again preparation of substratum

In order to prepare the interval extract of programming again, collect the interval cultured cells with standard method, by in the 10ml conical tube, coming washed cell in centrifugal 10 minutes under 4 ℃ with 500xg.Abandoning supernatant is resuspended in cell precipitation among the cold PBS that cumulative volume is 50ml.Under 4 ℃, with 500xg eccentric cell 10 minutes.Repeat this washing step, cell precipitation is resuspended in (20mMHEPES, pH8.2,5mMMgCl in the ice-cold interval cytolysis damping fluid of about 20 volumes ₂, 1mM DTT, 10 μ M press down the enzyme peptide, 10 μ M leupeptins, 10 μ M Pepstatin A, 10 μ M Trypsin inhibitor SBTIs and 100 μ M PMSF and 20 μ g/ml cytochalasin Bs selectively).Under 4 ℃, come sedimentation cell so that 800xg is centrifugal.Abandoning supernatant is resuspended in cell precipitation in the interval cytolysis damping fluid that is no more than 1 volume carefully.Culturing cell on ice 1 hour, make cell expansion.Carry out ultrasonication or carry out Dounce homogenate with glass mortar and pestle and come dissolved cell with most advanced and sophisticated ultrasonoscope.Dissolved cell is dissolved up at least 90% cell and nucleus, and this can evaluate by phase microscope.Being used to dissolve at least 90% cell and nuclear ultrasonic wave time will change according to the type of the cell that is used to prepare extract.

Cell lysates is placed in the centrifuge tube of 1.5ml, with desk centrifuge under 4 ℃ with 10,000-15, centrifugal 15 minutes of 000xg.From whizzer, take out test tube, be put on ice immediately.Collect supernatant liquor carefully with 200 μ l transfer pipet tips, will pool together, be placed on ice from the supernatant liquor of a plurality of pipes.This supernatant liquor is " interval is cytoplasmic " or " IS15 " extract.This cell extract can be distributed in the test tube on ice, every pipe 20 μ l, and freezing rapidly in liquid nitrogen immediately, preserve up to use down at-80 ℃.Alternately, cell extract is added on ice the ultracentrifugation pipe and (for example, is suitable for SW55 Ti rotor; Beckman).If desired, in test tube, cover mineral oil up to the top.With 200, the centrifugal extract of 000xg 3 hours is with the membrane vesicle that contains in the precipitation IS15 extract under 4 ℃.In the centrifugal later stage, discard mineral oil.Collect supernatant liquor carefully, if desired, pool together, be added in the cold 1.5ml test tube on ice.This supernatant liquor is called " IS200 " or " interval kytoplasm " extract.The extract mode that is used for IS15 as described is with this extract five equilibrium and freezing.

If desired, can be with other nucleus factor extract-enriched.For example, can be from the cell of the cell type in regeneration extract source or from the cell purification nucleus of any other cell type, and dissolve by ultrasonic wave as mentioned above.Under stirring, in containing NaCl that concentration is 0.15-800mM or KCl nucleus damping fluid, cultivate and extracted the nucleus factor in 10-60 minute.Centrifugal lysate precipitates insoluble composition.The supernatant liquor that dialysis contains the interested extraction factor removes NaCl or KC1.Nucleus extract five equilibrium and freezing preservation with dialysis.Before adding is used for the regenerated nucleus, this nucleus extract is added in the above-mentioned complete cell extract with various concentration.

Can also for example prepare the interval extract in ovocyte or the male sex-cell from sexual cell.For example, activate ovocyte as mentioned above, cultivate 5 hours to enter interval.Except in the dissolving damping fluid, saving the EDTA, as described in the embodiment 11, handle the ovocyte extract that ovocyte is used for II in mid-term then.The male sex-cell extract can be prepared as described in embodiment 11.

As substituting of cell extract, can also for example add in the solution in the damping fluid by one or more are natural or the factor of recombinating (for example nucleic acid or protein for example dna methyltransferase, histone deacetylase, histone, protamine, lamin, transcription factor, activator, repressor, Chromatin Remodeling albumen, somatomedin, interleukin, cytokine or other hormones).Preferably, one or more factor pair ovocytes or stem cell are special.

The cell of in substratum, programming again

The cell that to be changed processing thoroughly is with about 100-1, and the concentration of 000 cell/μ l is suspended in programme again substratum or the embodiment 11 described mitotic division of above-mentioned interval and programmes in one of substratum again.ATP generation structure and GTP are joined in the said extracted thing, cultivate down at 30-37 ℃ and reach 2 hours, from extract, transfer to the active cells nuclear uptake factor in the cell or karyomit(e) in conjunction with the factor to promote the factor.With the centrifugal cell of programming again of 800xg, by resuspended washing and centrifugal with 400xg in PBS.If desired, cell is resuspended in the substratum that contains 30% foetal calf serum (FCS), contain 2mM CaCl ₂The RPMI1640 of (adding in the 1M mother liquor by water preparation) or contain 2mM CaCl ₂α-MEM substratum in, in conventional cell culture incubator, cultivated 1-3 hour down in 37 ℃, cytolemma is sealed again.Washed cell in the warm substratum (10% FCS) of routine adopts the further culturing cell of type culture condition then.As described in example 17 above, Bian Cheng saturatingization cell can also be used to heredity and transfers in the ovocyte again, and does not need closing cell's film again.

The alternative method of the cell of saturatingization of programming again

Alternately, pair cell is changed processing thoroughly in the time of can be on being placed on cover glass, minimizes with the processing with pair cell, removing the centrifugal of pair cell, thereby makes the vigor maximization of cell.In 12 hole flat boards, allow grow into 50,000-100,000 cell/cover glass on the cover glass of poly-l-lysine bag quilt of the 16mm of cell (for example inoblast) in RPMI1640.Under the conventional gas condition under 37 ℃, saturatingization processing cell 50 minutes in the 200ng/ml streptolysin O of (Gibco-BRL) in no Ca2+HankShi balanced salt solution.If desired, can measure the ratio of the cell of being changed processing under these conditions thoroughly according to the picked-up of iodate third ingot.Streptolysin O is removed in suction; Be coated with the 80-100 μ l substratum of programming again on the cover glass; At 37 ℃ of following and CO ₂Culturing cell is 30 minutes to 1 hour in the gas.The substratum of programming again preferably contains ATP generation structure and the arbitrary ATP of 1mM, CTP, GTP and UTP.In order randomly to seal plasma membrane again, will contain 2mM CaCl ₂α-MEM substratum, contain the substratum of 20-30% foetal calf serum or contain 2mM CaCl ₂RPMI1640 add in the hole, 37 ℃ of following culturing cells 2 hours.Alternately, plasma membrane is not sealed again.

Streptolysin O is to saturatingization processing and the influence of closing cell's ratio again

For evaluation processing and closing cell's ratio again, carry out dosage and time titration (table 7) that streptolysin O is cultivated.In the latter stage that streptolysin O is handled, estimate the saturatingization processing of pair cell by the picked-up of 0.1 μ g/ml DNA dyestuff iodate, third ingot.Similarly in latter stage of sealing treatment again, the evaluation that each group cell is sealed again.

Table 7: fibroblasticization of ox processing that streptolysin O is handled and sealing again

To handle and be exposed to the evaluation of the fibroblastic viability of ox of mitotic division extract with streptolysin O

Carry out TUNEL and analyze, estimate with 0 or the 500ng/ml streptolysin O change thoroughly in the cell of processing and sealing again, perhaps change processing thoroughly with streptolysin O, be exposed to mitotic division extract 30 or the cell that also sealed again in 60 minutes in apoptosis.The TUNEL positive cell is that apoptotic cells (being necrocytosis) takes place.Data show, streptolysin O itself can apoptosis-induced (table 8).Analyze based on TUNEL, the cellular exposure that streptolysin O is handled in the mitotic division extract 60 minutes rather than 30 minutes makes apoptosis rate 10% (table 8) that rise.Based on these data, in extract, cultivate donorcells ratio cultivation in 30 minutes and be more preferably in 60 minutes.Carry out immunofluorescence analysis by pair cell, the result shows and cultivates the nuclear envelope disintegration caused most of checked cell in 30 minutes (about 90%, n〉100).

In addition, in extract, cultivate and damping fluid N or TL-HEPES and as the purifying cells that washs of the sucrose that is used for the chromatin transfer method in embodiment 11, described apoptosis (be respectively 2/34 and the 3/47TUNEL positive) can not take place

Table 8: streptolysin O and streptolysin O are added that the fibroblastic TUNEL of ox of extract-treated analyzes

The saturatingization method that selection is used for these cloning process is to handle 30 minutes with 500ng/ml SLO down at 38 ℃.It is to cultivate 2 hours containing α-MEM substratum that selection is used in the enclosure method again that the cell peripheral of programming again forms the intact cell film.

Use the alternative cell permeabilization treatment process of proteolytic enzyme

In an exemplary cell permeabilization treatment process that adopts proteolytic enzyme, allow cell (for example inoblast) in the 35mm flat board grow overnight to converging.Adopt conventional trypsinase-EDTA (0.5%-5.3mM) method to handle 5 minutes, take out inoblast from flat board.In PBS, wash inoblast, be resuspended in then among the 1ml TL Hepes.The 3mg/ml proteolytic enzyme that 1ml is dissolved among the TL Hepes adds (final protease concentration is 1.5mg/ml) in the 1ml cell suspending liquid to, at room temperature cultivates 1 minute.Washed cell in 10ml HBSS is resuspended among the 1ml HBSS and counting.With about 10 of minimum volume ⁵Individual cell is used for the mitotic division extract and cultivates.The MS-15 mitotic division extract of 40 μ l is added in the cell, placed 30 minutes down at 37 ℃.Washed cell in TLHepes is used for one of descriptive nuclear transplantation method of this paper then.

These having of cell signals, because after in the mitotic division extract, cultivating, concentrating of 70-80% cell generation nuclear envelope disintegration and ripe prochromosome shows that some composition from MPF in the extract (maturation promoting factor) or MPF has striden across cytolemma.This cell permeabilization method can with various proteolytic enzyme, for example trypsinase uses together, and using with undifferentiated cell with sexual cell, somatocyte, embryonic cell, fetal cell, one-tenth somatocyte, differentiation.

The formation of the ovocyte of reconstruct, activation, cultivation and transplanting

The microinjection of employing standard or electric fusion method, the cell of programming is again inserted or be fused in the acceptor ovocyte (referring to, for example United States Patent (USP) 4,994, and 384 and 5,945,577).For example, in the cell culture medium of standard, when existing or lack sucrose (for example 2.5% sucrose), cell is placed near ovocyte, and cell can be imported in the injection suction pipe.This suction pipe of pressure-vaccum takes out the kytoplasm composition several times with dissolved cell from nucleus then, then it is expelled in the nucleus.Activate ovocyte, the cultivation of reconstruct then, and adopt example those standard method as described in example 11 above to be transplanted in the parent acceptor Mammals, to produce clone's Mammals.

Embodiment 13: adopt chromatin transfer and streptolysin O to transplant to carry out the evidence that nucleus is programmed again more completely

Described in embodiment 8-10, in traditional core transplanting protokaryon stage embryo incomplete nucleus takes place and reinvent and programming again.This discovery obtains that Lamin A/C assembles and the confirmation of excessive NuMA immunofluorescence label in the nuclear envelope of nuclear transplantation protokaryon embryo.Adopt cell permeabilization processing described in chromatin transfer method described in the embodiment 11 and the embodiment 12 and the more complete nucleus of reprogramming method (being also referred to as SLOT) realization to programme again.

Especially, and compare with the clone embryos of traditional cloning process production, the embryo that cloning process of the present invention generates has the protein expression mode that is similar to embryo in vitro fertilization more.Shown in embodiment 8-10, the Lamin A/C albumen that the embryo that chromatin shifts expresses than traditional core transplanting embryo still less.Lamin A/C is the special composition of the somatocyte in the lamin, expresses naturally in noble cells, and does not express in the embryo.Because lamin of being reported and the interaction between transcription factor, chromatin albumen and the DNA, the expression of possible Lamin A/C in traditional nuclear transfer embryo promotes to be specific to somatic protein expression, and these are specific to somatic protein and do not expect for fetal development.Therefore, the somatocyte specific proteins of not expecting of chromatin transfer embryo of the present invention expression may be than traditional nuclear transfer embryo still less.Alternately, chromatin shifts the expression pattern that the embryo has NuMA, and NuMA is the main component of nuclear matrix, the regulation and control that participation is transcribed, and chromatin shifts the embryo and more is similar to embryo in vitro fertilization than traditional core transplanting embryo.This result shows that also the embryo that chromatin shifts is more effectively programmed than traditional nuclear transfer embryo again.

Evaluation to the external caryorrhexis of ox inoblast nuclear

All the time support the disintegration (accompanying drawing 33) of the input that the inoblast of about 80% purifying is examined from the extract of mitotic ox inoblast preparation.From the extract (promptly from the progamous extract that is stuck in II ovocyte in mid-term naturally) of II ovocyte in mid-term also sustenticular cell caryorrhexis (nucleus of disintegration 75% in 30 minutes) successfully.

The expression of the following mark in the chromatin (accompanying drawing 34C) of the interphase nuclei (accompanying drawing 34A) that detection imports, the chromatin (accompanying drawing 34B) that from the nuclear of cultured cells MS15 mitotic division extract, obtains, acquisition from the nuclear of cultured cells the ovocyte extract: lamin B acceptor (LBR), it is the integral protein (membrane marker) of inner nuclear membrane; Lamin B, it is the omnipresence composition of lamin; Lamin A/C, it is to exist only in noble cells and the somatocyte specific component that is not present in the lamin among the embryo; NuMA, the main component of nuclear matrix and DNA.Somatocyte kytoplasm MS15 and ovocyte MS15 extract cause that all lamin B, Lamin A/C, LBR and NuMA are dissolved in about 100% the chromatin unit of being detected (accompanying drawing 34B and 34C).As expected, as before viewed in mitotic people's cell, AKAP95 keeps related with karyomit(e) (Collas etc., J.Cell Biol.147:1167-1180,1999).This result also appears in the nuclear transplantation in cattle embryo of the ripe prochromatin enriching stage of embodiment 8.Mitotic division extract and ovocyte extract promote aspect the nuclear envelope dissolving as if all the same effective with complete ovocyte, and with used method, promptly traditional nuclear transplantation, nuclear injection (NI) or chromatin shift irrelevant (accompanying drawing 35)

By the protokaryon embryo of chromatin transfer production and from the protokaryon of nuclear transplant and the comparison between the nuclear embryonal vaccination

In order to produce the embryo that chromatin shifts, after maturation, made the ovocyte stoning of maturation in vitro in about 18-20 hour.Will from interval bovine fetal fibroblast nucleus in MS15 mitotic division extract, cultivate, as described herein from the ox fetal cell preparation this extract.The nuclear envelope disintegration takes place after and chromatin concentrate finish before, from extract, separate chromatin.Especially, when having the chromatin of 50-60% to be concentrated approximately with the karyomit(e) enriched level of interval (being considered as 0% concentrates) and mitotic maximum karyomit(e) enriched level (being considered as 100% concentrates), separation chromatin.In this stage, be difficult to distinguish the individual chromosome in each chromatin, and chromatinic edge has irregular shape.To from the mitotic division extract, place TL HEPES droplet with enucleation oocyte by isolating chromatin with 2.5% sucrose.Adding sucrose minimizes the destruction of follow-up injection operation to ovocyte.(Fishers NY) is expelled to chromatin in the ovocyte (under the amplitude of 70V with 2Hz frequency processing 75 microseconds) with the inclined-plane microinjection suction pipe with Burleigh Piezo Drill.Usually adopt a plurality of pulses, for example 2,3,4 or 5 pulses make syringe needle be enough to penetrate ovocyte and inject.After injection, in the TL HEPES that contains sucrose of serial dilution, wash ovocyte, so that osmotic shock is minimized.(promptly placed 28-30 hour in maturation medium after collecting from ovary in 28-30 hour ripe back, simultaneously after injecting chromatin at least 2 hours), with the ovocyte of reconstruct and contrast 4 minutes (the Cal Biochem of Calcium ionophore (5 μ M) activation that are used for the parthenogenesis growth, San Diego, CA) and be dissolved in ACM substratum (100mM NaCl, 3mM KCl, 0.27mM CaCl ₂, 25mM NaHCO ₃, 1mM Sodium.alpha.-hydroxypropionate, 0.4mM pyruvate salt, 1mM L-glutaminate, 3mg/ml BSA (FAF), 1%BME amino acid and 1%MEM non-essential amino acid (Sigma)) 10 μ g/ml cycloheximides and 2.5 μ g/ml cytochalasin D (Sigma) in 5 hours (Liu etc. of activation as discussed previously, Mol.Reprod.Dev.49:298-307,1998).After the activation, washing ovum 5 times is placed in the 4 hole tissue culture plate mineral oil (Sigma) that has covered the 0.3ml embryo testing, that contain l cell and 0.5ml embryo culture medium and is cultivated.25-50 piece of embryo is placed in each hole, under 38.5 ℃ at 5%CO ₂Cultivate under the atmosphere surrounding.If desired, with calcium (for example, the CaCl of about 0.5,1.0,1.5,2.0,2.5,3,3.5,5mM or greater concn ₂) add in the substratum, keep about 0.5,1.0,1.5,2.0,2.5,3.0 or more hours, to promote that ovocyte is sealed again after injection.Because when ovocyte being implanted in the acceptor Mammals with standard method described herein, complete cytolemma may improve the survival rate of the ovocyte of sealing again around ovocyte.

Shift the embryo as the above-mentioned chromatin that carries out, the product nucleus embryonal vaccination is except the interval bovine fetal fibroblast nuclear that will not cultivate in extract is expelled in the ovocyte rather than in the chromatin.Adopt the produced in conventional processes nuclear transfer embryo of describing among the embodiment 8.

The protokaryon that nuclear transplantation as expected, nuclear injection and chromatin shift is ressembled lamin B (accompanying drawing 36A, red-label) and AKAP95 (accompanying drawing 36B, red-label).Nuclear transplantation and nuclear injection protokaryon be assembly cell-specific composition Lamin A/C (accompanying drawing 36A, Green Marker) also, and be consistent with the result of the above-mentioned nuclear transfer embryo of being reported.But the protokaryon that chromatin shifts the parthenote of protokaryon and contrast does not assemble Lamin A/C (accompanying drawing 36A).Shift or parthenote protokaryon different (accompanying drawing 36B, Green Markers) with most of chromatin, the nuclear transplantation protokaryon also contains NuMA (Green Marker).As above report a certain proportion of parthenogenesis somatic cell nuclear and the low-level NuMA of chromatin transitional cell nuclear assembling.

The external decomposition of nucleus after chromatin shifts, chromatin are shifted and are created in the protokaryon that is similar to contrast parthenote protokaryon on the form.On the contrary, nuclear transplantation and nuclear injection protokaryon have somatocyte specific component (Lamin A/C and NuMA mark) widely.This result shows, after traditional core is transplanted or examined injection operation incomplete nucleus takes place and reinvents.As mentioned above, detected Lamin A/C derives from the lamin of transcribing again in the protokaryon stage in nuclear transplantation and nuclear injection protokaryon.Because lamin and possible NuMA relate to transcriptional control and human diseases, in conventional nuclear transplantation protokaryon, continue to exist Lamin A/C to represent unsuitable functional programming again.The inventor concludes that cell in vitro nuclear decomposition and chromatin shift to produce than traditional core and transplants or the more normal protokaryon of nuclear injection.

Use and programme chromatin again or change the cloning efficiency of the cell of processing thoroughly as donor source

As described in Example 12, developed the cloning process of a kind of being marked as " SLOT ", it comprises saturatingization of inducing former generation bovine fetal fibroblast with streptolysin O, with the cellular exposure changed thoroughly in the substratum of programming again (for example mitotic division extract) 30 minutes, selectively cultivate and seal inoblast again, chromatin is transferred in the ovocyte with the cell fusion method of standard with 2mM calcium.

For this cloning process, with one bottle of streptolysin O (Sigma S-5265; 25,000 units are kept under 4 ℃ with the storage powder form) be dissolved in the 400 μ l water thorough mixing.Entire contents is transferred in the conical tube of 15ml, added the water of 3.6ml then, mix by vibration.The aliquots containig of 10 μ l is freezing down at-20 ℃ with the mother liquid concentration of 0.062U/ μ l.At room temperature, cell (about 100,000) is suspended among the 100 μ l HBSS (Gibco BRL, catalog number 14170-120).These cells converge, and therefore about 80-85% cell is in the G1 phase, and most other cells are in the S phase.Add streptolysin O mother liquor (5 μ l) (that is, the ultimate density of 500ng/ml or 0.3U/ μ l), the incubation mixture is 25 minutes in 38 ℃ of water-baths.In the incubation process, pat test tube 2-3 time gently to guarantee that cell still is in suspended state.Add room temperature PBS (200 μ l), by soft piping and druming with thorough mixing.At room temperature, in desk centrifuge with 5,000rpm eccentric cell 5 minutes.Discard whole supernatant liquors.In this stage, precipitate lessly, can not clearly be seen.Add the mitotic division extract (40 μ l " MS15 ") that contains the ATP generation structure, thorough mixing.In the process of eccentric cell, by 1 bottle, the 40 μ l extracts that thaw, and add 1.2 μ l ATP generation structures, thorough mixing, at room temperature incubation prepares extract.This mitotic division extract is with to be used to produce chromatinic extract in above-mentioned chapters and sections identical.The incubation mixture is 30 minutes in 38 ℃ water-bath, and pats test tube once in a while gently.The substratum of sealing again (RM, 500 μ l) that adds room temperature (has added the 1M mother liquor and has made CaCl ₂Complete α-MEM[Bio-Whittaker for 2mM]).Open wide test tube, at CO ₂Incubation is 2 hours in the incubator, pats test tube once in a while and is in suspended state to guarantee cell.At room temperature, in desk centrifuge with 5,000rpm eccentric cell 5 minutes.Cell precipitation is resuspended among the TLHEPES (Bio-Whittaker, catalog number 04-616F) under the 100 μ l room temperatures, and adds 900 μ l TLHEPES again.Adopt standard method to carry out nuclear transplantation, as described in the chapters and sections of the relevant chromatin transfer in front, activate ovocyte, and be implanted in the acceptor mammalian body.

Adopt the embryo's of this SLOT method and chromatin transfer method of the present invention production growth to be summarized in the table 7.Compare with traditional nuclear transfer embryo, the SLOT fetal development is arrived the blastaea stage and can be lacked a little.The difference that SLOT and nuclear transfer embryo are grown the blastaea stage may be because the ratio of the cell that is in the cell cycle G1 phase that uses during nuclear transplantation is higher than SLOT.The embryo's that chromatin shifts survival rate is lower, and chromatin shifts and is considered to a kind of invasive method.

For nuclear transplantation and SLOT embryo, has comparability (table 9) in 40 days pregnancy rate of gestation.The SLOT embryo often is higher than the nuclear transfer embryo of using produced in conventional processes pregnant 40-60 days survival rate.

Table 9: the developmental state of the ox embryo cloning that transplanting of chromatin transition kernel and SLOT produce

	Transplant number	Survival number (%)	PN number of stages (%)	Spilting of an egg number (%)
	Transplant number	Survival number (%)	PN number of stages (%)	Spilting of an egg number (%)	CT	1503	736(49)	355(23.5)	81(5.3)
SLOT	1884	1802(97)	ND	575(30.5)	CT	1503	736(49)	355(23.5)	81(5.3)
SLOT	1884	1802(97)	ND	575(30.5)	Nuclear transplantation	1821	1682(92)	ND	764(41.9)

	Blastaea number (%)	40 days quantity (%) of gestation	Quantity/the sum (%) of survival in 40-60 days
	Blastaea number (%)	40 days quantity (%) of gestation	Quantity/the sum (%) of survival in 40-60 days	CT
	3	0	ND	CT
	3	0	ND	SLOT	156(8.3)	24/65(37)	7/10(70)
Nuclear transplantation	235(12.9)	39/103(36)	8/16(50)	SLOT	156(8.3)	24/65(37)	7/10(70)

As noted above, can make and ovocyte sealing again before being implanted to the acceptor Mammals improve the survival rate that chromatin shifts the embryo by in calcium ion, cultivating the ovocyte of reconstruct.Can by reduce that cell takes out from substratum and and the ovocyte fusion between time quantum improve SLOT embryo's survival rate.For example, can reduce the cultivation in streptolysin O, the time span of in the substratum of programming again, cultivating and/or in the sealing substratum, cultivating.Especially, sealing again that the time of cultivating in the substratum can be reduced to about 1 hour or still less.Can improve enclosed more as mentioned above having the 2mM calcium ion or when having greater concn calcium ion (for example, pact-2.5,3.0,3.5,4.0,4.5,5.0 or 6.0mM calcium ion), shortening close process time again.By shortening the time quantum of the processing cell before merging with ovocyte, cell enters the S phase and begins the possibility of dna replication dna littler, enters the S phase and begins the survival rate that dna replication dna can reduce the reconstruct ovocyte.

Embodiment 14: the evidence that adopts chromatin to shift to carry out nucleus more completely to programme again

As discussing among the embodiment 11, develop a kind of strategy and strengthen reinventing of donorcells nuclear, promote the inhibition of somatic cell gene, produce embryo with the former nuclear structure that is similar to the fertilization zygote.In the specific embodiment of this normal dyeing matter transfer method, isolating complete ox inoblast nucleus is cultivated in the fibroblastic tenuigenin extract of the mitotic division ox that contains the ATP generation structure, and the ATP generation structure is used to drive nuclear assembling.

Be used for donor nuclei is converted to the chromatinic mitotic division extract of programming again in order to generate, handled about 18 hours with 0.5 μ g/ml Luo Keda azoles, with the inoblast synchronization at m period, the method of shaking off by m period is gathered in the crops, and in ice-cold PBS, wash 2 times, at ice-cold cytolysis damping fluid (20mM Hepes, pH8.2,5mM MgCl ₂, 10mMEDTA, 1mM DTT and proteinase inhibitor) washing 1 time in (Collas etc., J.Cell Biol.147:1167-1179,1999).The cell that compresses is resuspended in the 1 volume cytolysis damping fluid, expanded 1 hour, and carry out Dounce homogenate with sizeable pestle on ice on ice, dissolved up to cell.With 15, the centrifugal lysate of 000xg 15 minutes is collected supernatant liquor (mitotic division extract) under 4 ℃, and five equilibrium is also freezing rapidly in liquid nitrogen, is stored under-80 ℃.

In order to produce donor chromatin, gather in the crops the not synchronized inoblast that converges, wash, be resuspended in ice-cold hypotonic nucleus dissociating buffer (10mMHepes, pH7.5, the 2mm MgCl of about 20 volumes ₂, 25mM KCl, 1mM DTT and proteinase inhibitor mixture) in (Collas etc., J.Cell Biol.147:1167-1179,1999).After placing 1 hour on ice, carry out Dounce homogenate with sizeable pestle pair cell.It is 250mM that adding 2M sucrose mother liquor makes sucrose concentration, examines 10 minutes with the 400xg centrifugation cell under 4 ℃.At nucleus dissociating buffer (damping fluid same as described above, but sucrose concentration is 250mM) middle washed cell nuclear, and use immediately or be chilled in (Collas etc., J.Cell Biol.147:1167-1179,1999) in nucleus dissociating buffer/70% glycerine.

For programming again, contain ATP generation structure (1mM ATP at 40 μ l, 10mM phosphocreatine and 25 μ g/ml sarcosine kinases) the mitotic division extract in the inoblast nucleus of culture of isolated, in 38 ℃ of water-baths, cultivated 30 minutes with 4,000 nucleus/μ l.Concentrate by phase microscope monitoring nuclear envelope disintegration and chromatin.Cultivating latter stage, compound of reaction is diluted with the TLHepes that 500 μ l contain 2.5% sucrose, by with 2, chromatin was gathered in the crops in 000x g centrifugation in 5 minutes.Chromatin is resuspended in the TL Hepes/ sucrose, transfers in the TLHepes/ sucrose under the mineral oil with enucleation oocyte.(Fishers, NY) (2Hz, 2 μ s 70V) are expelled to single chromatin in the ovocyte of maturation in vitro, and ovocyte has used the microinjection suction pipe on inclined-plane with the 20hpm stoning with Burleigh Piezo Drill.After the injection, in the serial dilution of the sucrose that is dissolved in Hepes, wash ovocyte, osmotic shock is minimized, and cultivate.If be used for NT, then activate the ovocyte and the parthenogenesis contrast of reconstruct, and cultivate ((Kasinathan etc., Nat.Biotechnol.19:1176-1178,2001 and Kasinathan etc., Biol.Reprod.64:1487-1493,2001) with 28hpm.For nuclear injection (NI), the inoblast nucleus of purifying is exposed in the cytolysis damping fluid, rather than in the mitotic division extract, if be used for CT, then with nuclear injection in non-nucleus egg mother cell.

Because programming again, Lamin A/C, lamin B and NuMA are easy to decompose, and AKAP95 still combines (accompanying drawing 42A) with spissated karyomit(e).Apoptosis does not take place in the TUNEL analysis revealed in the extract.Personnel selection Hela nucleus and mitotic division extract carry out similar nucleus slaking test, the result show spissated chromatin can sustenticular cell nuclear reconstruct (Steen etc., J.Cell Biol.150:1251-1562,2000) and transcribing in interval tenuigenin.Therefore, produced can the nuclear condensed chromatin of regenerating functional in the cell in vitro nuclear decomposition.Reclaim spissated fibroblastic chromatin by precipitation, and single chromatin is expelled in the acceptor ovocyte of stoning.After reclaiming culture, as being used for the described method of NT ovocyte, embodiment 9 activates ovocyte.In order the artefact who generates by the nucleus operation to be regulated and control, has also injected the intact cell nuclear that only is exposed to extract.

The result of CT and contrast nuclear injection (NI) is presented among the accompanying drawing 42A-42D.The protokaryon that NI produces as the NT protokaryon, contains Lamin A/C and B, NuMA and AKAP95 (accompanying drawing 42B and 42C).CT cause more than 80% in injection and the embryo of survival in activating form PN (be about〉2,000) by 50% of injection ovocyte, n.But the CT protokaryon has shown undetectable Lamin A/C, and anti-NuMA is immune labeled to have reduced by 3 times (accompanying drawing 42B and 42C) than NT and NI protokaryon.This pattern is similar to gynecogenic protokaryon.Compare with the NT protokaryon, in the CT protokaryon, the possibility of using Triton X-100/DNA enzyme I/NaCI to extract aforesaid AKAP95 and DNA has improved 8 times, shows the beneficial effect (accompanying drawing 42D) of CT to the accessibility of protokaryon AKAP95 grappling and DNA enzyme I.Heat up in a steamer owing to the chromatin in conjunction with AKAP95 is divided into to separate with the chromatin of originally transcribing inhibition (low acetylizad), this result shows that CT strengthens formation euchromosome when PN assembles.

In be carried out to the process that fibrocyte donorcells nuclear reinvents by the NT that implements abreast and CT method, detect the kinetics of the conjugated protein TBP of TATA.In fact, for all genes, TBP promotes the assembling (Sharp etc., Cell 68:819-821,1992) of the mechanism of generally transcribing.In the fibroblastic nucleus of donor, TBP locatees (accompanying drawing 43A) altogether with AKAP95 and DNA.Contain TBP than 5 times of spissated dyeing heights in the mitotic division extract, (the accompanying drawing 43B) shown in the fluorescence intensity ratio of TBP/AKAP95 and TBP/DNA at the PCC karyomit(e) that obtains behind the NT.Similar in TPB mark intensity in external spissated karyomit(e) and the mitotic division inoblast.In the PN stage, in the CT embryo, almost can not detect TBP, but in NT and NI embryo, have very strong immune response activity (accompanying drawing 43A).Protokaryon TBP among the NT embryo is the somatocyte source, because stronger TBP mark is kept in the inhibition of transcribing or translating.Verified, when being reacted to interval, the protokaryon TBP in the mouse increases (Worrad etc., Development 120:2347-2357,1994).But the similarity of kinetics in NT that rebuilds and CT embryo that is formed PN by PCC or external spissated karyomit(e) show, it is not that cell cycle phase owing to higher level causes that the TBP concentration in the NT protokaryon raises.Therefore, the external decomposition of donorcells nuclear promotes that TBP separates from karyomit(e), makes the CT protokaryon that generates contain the TBP than low 10 times of the NT protokaryon of identical reconstruction stage.

Therefore, 5 the structural and functional marks of programming not exclusively again by the NT method comprise that concentration rising, AKAP95 and the DNA of protokaryon assembling, protokaryon NuMA and the TBP of Lamin A/C reduce the susceptibility of extracting with washing agent, DNA enzyme and salt.On the contrary, the Type B lamin forms for correct nucleus again and cell survival is crucial (Steen etc., J.Cell Biol.153:621-626,2001), though can not determine the Type B lamin that is assembled is somatocyte source (and therefore being practiced shooting again), still as if female parent is originated, and the Type B lamin can assembling normally in the NT protokaryon.The LA gene still has activity in the NT protokaryon, cause the assembling of the A type lamin that noble cells is special.By chromatin and transcribe interaction between mechanism, the nucleus lamin is considered to participate in transcriptional control (Cohen etc., Trends Biochem.Sci.26:41-47,2001); Therefore, the assembling most probable of a correct cover lamin is crucial for former kernel function correct in the NT cell.

In the method for the invention, somatic nucleus composition is dispersed in the extract, and does not contact with the kytoplasm of ovocyte usually.This has prevented that the special molecule of somatocyte from practicing shooting to developmental nucleus again, and somatocyte special molecular such as Type B lamin, its composition are different from the composition (Cohen etc., Trends Biochem.Sci.26:41-47,2001) of maternal lamin.External chromatin concentrates and also may promote for example release of Chromatin Remodeling enzyme (Sif etc., Genes Devel.12:2842-2851,1998) and transcription factor of dna binding factor, thus the genome of the potential inhibition somatocyte composition of " peeling off " donor.Especially, TBP removes the somatocyte specific gene inactivation that may cause in the regenerated CT protokaryon, and the transcriptional activity of the low male pronucleus of after fertilization is doubled (Poccia etc., Trends Biochem.Sci.17:223-227,1992).

Removing the factor from donorcells nuclear means and is loaded on the chromatin and reinvents in the physiological protokaryon being convenient to maternal composition.CT has improved AKAP95 and the DNA susceptibility to nuclease and salt.The DNA major part of DNA enzyme I resistance is a Transcriptional Silencing; Therefore, may reduce not exclusively reinventing of AKAP95 grappling by NT and to have grown the expression of going up important function of gene, for example with placenta is grown, the later stage pregnancy maintenance is relevant with the postnatal viability of cloned animal gene.

The following feature of CT ovocyte shows that the chromatinic nuclear transplantation of cultivating has improved the functional character of the regeneration ovocyte that generates significantly in the mitotic division extract.That expresses in CT protokaryon (chromatin being imported in the ovocyte protokaryon that the back forms) hangs down 3 times by the nuclear matrix protein NuMA of somatocyte donorcells high level expression in than NT protokaryon.This result shows that the NuMA content in the CT ovocyte is compared the NuMA content that more is similar in the natural ovocyte with the NT ovocyte.The CT protokaryon is the transcription factor commonly used of low 10 times of expression ratio NT protokaryon also.Be inactivated by the gene of in the mitotic division extract, cultivating in the CT ovocyte that the TBP that removes like this may cause generating from donor chromatin that do not expect, that somatocyte is special.Lamin A/C is a kind of nuclear envelope albumen, is specific to the cell of differentiation, is not detected in vitro fertilization or parthenogenesis activated ovocyte, also detects in the CT protokaryon less than this albumen, but can detect in the NT protokaryon.Because lamin may be regulated and transcribe, lack detectable Lamin A/C in the CT protokaryon and may in the CT ovocyte, produce than adjusting more appropriate in the NT ovocyte to transcribing.The structural albumin A KAP95 at nuclear matrix chromatin interface enrichment in the chromatin of transcribing inhibition (low acetylize); in protokaryon, measure AKAP95 and the susceptibility of DNA, to characterize the grappling and the accessibility that characterize DNA in protokaryon of AKAP95 to DNA to extracting with washing agent, DNA enzyme and salt.Compare with the NT protokaryon, the extractibility in the CT protokaryon has improved 8 times, has shown in the mitotic division extract to cultivate the beneficial effect that donor chromatin is reinvented AKAP95 grappling and donor dna form.Because AKAP95 and DNA enzyme I resistance DNA are with to transcribe DNA inhibition or reticent relevant, AKAP95 and DNA raise to the susceptibility of nuclease, salt and washing agent, show that the CT ovocyte may increase to express the upward expression of important function of gene of growth.

Expection is endorsed for the donorcells that comes to have in the comfortable prion gene cell of one or more sudden changes and is obtained similar result.

Embodiment 15: shift the evidence of realizing that nucleus is more completely programmed again with streptolysin O

Use SLOT to cultivate in the mitotic division extract in the process of the cell of being changed processing thoroughly, chromatin is programmed and also is shown among accompanying drawing 45A and the 45B.In this test, at the ovocyte of producing with SLOT with in the ovocyte of the nucleus of in extract (" NT "), not cultivating by the production of traditional core implantation method, the relatively expression of the nuclear matrix protein NuMA of high level expression and the expression that is specific to the nuclear envelope albumen Lamin A/C of noble cells in the somatocyte donorcells.Activate back 14 hours, NuMA and Lamin A/C that SLOT protokaryon (promptly will contain the protokaryon that chromatinic donorcells imports to back formation in the ovocyte) is expressed lack (n=15-20 embryo/mark, 3 repetitions) than the NT protokaryon significantly.These results show that regenerated SLOT ovocyte is programmed than NT ovocyte more again, and more are similar to the ovocyte of natural fertilization.Because lamin may be regulated and transcribe, significantly the Lamin A/C of lower aq may produce in the SLOT ovocyte than the regulation and control to transcribing more suitable in the NT ovocyte in the SLOT protokaryon.

Except the expression that in the ovocyte that generates, reduces expecting factor not, compare with traditional nuclear transplantation method, SLOT has improved the developmental potency (table 10) of the fetus that generates.Therefore, the structural and functional difference in many SLOT donorcellses has improved the ability of regenerated ovocyte formation non-human embryo and non-human mammal in fact.

Table 10: the ox embryo's (" NT ") that the growth that apparatus has an ox embryo (" SLOT ") that the donorcells of chromatinicization produces and the nucleolate donorcells of apparatus are produced growth comparison

	Transplant quantity	Acceptor quantity		40 days quantity (%) of gestation	90 days quantity (%) of gestation
	Transplant quantity	Acceptor quantity		40 days quantity (%) of gestation	90 days quantity (%) of gestation	SLOT	1955	59	29/59(49)	14/59(24)
NT	1885	95	44/95(46)	16/95(17)		SLOT	1955	59	29/59(49)	14/59(24)

Embodiment 16: adopt streptolysin O to shift (SLOT) and carry out the exemplary evidence that nucleus is more completely programmed again

As described in

embodiment

12 and 15, developed a kind of new external nuclear and reinvented system.This comprising of system processing donorcells, induce in the mitotic cell extract that chromatin concentrates, the cell of washing saturatingization processing is with removal dissolved nucleus factor in the chromatin concentration process.Somatic nuclear transfer embryo is opposite with being similar to, and the expression pattern of several marks that ox chromatin transfer embryo's protokaryon has approaches the expression pattern of fetal tissues.Produced the calf of 8 health with this chromatin transfering system.Chromatin shifts has the trend that survival is extended to mature, low big fetus incidence and significantly improves birth back survival rate.This result shows before transplanting successfully somatocyte donorcells nuclear is operated.As further describing hereinafter, somatic cell nuclear decomposes in the mitotic division extract, then spissated chromatin is transferred in the ovocyte, has improved nucleus and has reinvented, and demonstrates the developmental potency and the vitality that have improved the clone.If desired, this method can be used to the further characterize cells nuclear mechanism of programming again.

The chromatin transition strategy

In order to eliminate in protokaryon NT embryo the defective of determining, with fibroblastic nuclear transplantation before the acceptor ovocyte, it is carried out manipulation in vitro.Summarized the SLOT system in the accompanying drawing 48.For production mitotic division extract, be processed into fibrocyte 18 hours with 1 μ g/ml Luo Keda azoles, with its synchronization at m period, come harvested cell by the mitotic side of shaking off, washing is 2 times in the physiological saline of phosphoric acid buffer, at cytolysis damping fluid (20mMHepes, pH8.2,5mM MgCl ₂, 10mM EDTA, 1mM DTT and proteinase inhibitor) in washing 1 time.Precipitated cell is resuspended in the ice-cold cytolysis damping fluid of 1 volume,, carries out Dounce homogenate with sizeable glass pestle to expand on ice 1 hour.Under 4 ℃ with 15, the centrifugal lysate of 000x g 15 minutes, five equilibrium supernatant liquor (mitotic division extract), freezing in liquid nitrogen, and be stored under-80 ℃.Use fresh or the refrigerated extract, do not having marked difference aspect the nucleus disintegration efficient.

At no Ca ²⁺/ Mg ²⁺HankShi balanced salt solution (HBSS) in the washing from the bovine fetal fibroblast that converges culture, by in about 38.5 ℃ of water-baths, at the 31.2U streptolysin O (SLO that is dissolved in 100 μ l HBSS; Sigma) cultivating 100,000 cells in the suspension changes thoroughly.By the not picked-up evaluation processing of the DNA dyestuff propidium iodide of permeate through cell membranes (0.1 μ g/ml).Precipitation is changed the inoblast of processing thoroughly, washing, under about 38.5 ℃, contain in the mitotic division extract of ATP production system (1mM ATP, 10mM phosphocreatine and 25 μ g/ml sarcosine kinases) at 40 μ l and to cultivate 30-45 minute, promote nucleus to decompose and remove the nucleus composition.Chromatinic concentrated with 0.1 μ g/ml Hoechst, 33342 mark aliquots containigs to monitor.After the cultivation, from extract, be recycled into fibrocyte by precipitation, and washing.Contain 2mM CaCl with 500 μ l ₂α MEM/10% foetal calf serum (Gibco-BRL) diluted reaction mixture, with closing membrane again, at 38.5 ℃ of following culturing cells 2 hours (Hakelien etc., Nat.Biotechnol.20:460-466,2002).Monitor sealing again by the picked-up of iodate third ingot.The cell that is sealed again and the ovocyte of maturation in vitro merge, and this ovocyte is with the 20hpm stoning; Activate ovocyte with 28hpm, cultivate the embryo at NT described as embodiment 10.

External the SLOT embryo culture is arrived the blastaea stage, transplant 2 pieces of embryos for every recipient female animal.By ultrasonic inspection method monitoring gestation, carry out the C-tomoscan.In postnatal 24 hours, calf is marked by the animal doctor.

The disintegration of fibroblastic nucleus in the mitotic division extract

15 of the fibroblastic lysate of the own silk division ox of mitotic division extract origin, 000x g centrifugal supernatant liquor is formed, and contains the ATP generation structure.This extract can be not apoptosis-induced, judges (accompanying drawing 49A) by the protein hydrolysis and the fibroblastic dna break feature of apoptosis that do not have poly (ADP) ribosyl polysaccharase (PARP).Therefore, this extract is suitable for promoting reinventing of somatic cell nuclear.

The karyomit(e) that this extract brings out the ATP dependence concentrates, removes TBP (accompanying drawing 49B) from the decomposition of chromatinic A/C and Type B lamin and from chromatin, and the decomposition of A/C and Type B lamin is judged by the kytoplasm mark of these lamins.After from the mitotic division extract, reclaiming,, confirmed these incidents (accompanying drawing 49C to the immunoblotting assay that the condensed chromatin from fibroblastic purifying carries out; Compare swimming lane 1 and 3).In this test, A kinases anchorin AKAP95 is used as still the mark of the nucleus composition relevant with condensation, and this usually occurs in m period (Collas etc., J.Cell Biol.147:1167-1180,1999).The protein that histone H 4 is used as in the gel loads contrast (accompanying drawing 49C).Decomposition from chromatinic lamin and TBP depends on ATP regenerating system (accompanying drawing 49B and 49C, swimming lane 3 and 4), as taking place in the mitotic cell (accompanying drawing 49C, swimming lane 2).In addition, the inoblast (with respect to isolating chromatin) that complete quilt after being exposed in the mitotic division extract is changed thoroughly carries out immunoblotting assay, the result shows, part dissolved Lamin A/C and all detectable TBP are eliminated from cell and/or by proteolysis (accompanying drawing 49C, swimming lane 6).At last, all contain the ATP regeneration system from fibroblastic reference extract of interval (accompanying drawing 49C, swimming lane 5) or simple cytolysis damping fluid, all can not promote nucleus to decompose, show that the nucleus disintegration is that ATP relies on, and be specific to the mitotic division extract.Be exposed to the mitotic division extract and use CaCl ₂The inoblast of Feng Bi saturatingization can be cultivated number generation again, shows that at film changing processing thoroughly, cultivating in extract that the cell of being changed processing thoroughly and film seal again all is feasible method.

In the nuclear sign that shifts by chromatin among the embryo who produces

Feng Bi inoblast is taken place with the efficient the same with the cell of not changed processing thoroughly (surpassing 70%) with the fusion of acceptor ovocyte again.When importing to ovocyte, donor chromatin be not conc forms (accompanying drawing 50A, SLOT).On the contrary, remain in the fibroblastic chromatin that is used for NT 30 minutes after fusion depolymerization (accompanying drawing 50A, NT).Therefore, before transferring to ovocyte, use CaCl ₂Seal inoblast again, can in donorcells, not promote nucleus regeneration through the mitotic division extract-treated.Judge by immunofluorescence analysis nuclear lamina (lamina) and internal layer cell membrane protein, after fusion, around SLOT embryo's condensed chromatin, lack nuclear envelope immediately and supported this observations.

Immune labeled lamin and TBP in SLOT and NT embryo's nucleus, and these marks are shown among accompanying drawing 50B and the 50C with respect to the immune labeled intensity of DNA fluorescence intensity.The lamin B mark intensity in nuclear week similar with in SLOT and NT protokaryon.But, different with the NT protokaryon significantly, at protokaryon with to 8-16 cell stage at least, in the SLOT embryo, detect less than Lamin A/C (accompanying drawing 50B).Compare with the NT protokaryon, the SLOT protokaryon also demonstrates 4 times (accompanying drawing 50C) of TBP mark decline.Verified, growing in the process of interval the concentration of protokaryon TBP rising (Worrad etc., Development 120:2347-2357,1994) in the mouse.But owing to forms the kinetics of protokaryon similar with in NT and SLOT embryo by PCC or external spissated chromatin, though be not to get rid of definitely, the rising of TBP concentration can not be owing to being in the more cell cycle of high-stage in the NT protokaryon.TBP has reduced (accompanying drawing 50D) more than 2 times to the resistance of extracting with 1% Triton X-100,1mg/ml DNA enzyme I and 0.3M NaCl, shows that TBP combines insecure with intracellular aglucon.Similarly, in the SLOT protokaryon, DNA has reduced by 4 times nearly to the resistance of DNA enzyme I, shows that SLOT helps setting up comparatively loose chromatin Structure (accompanying drawing 50D) in protokaryon.Therefore, fibroblastic nucleus decomposes in the mitotic division extract, then spissated chromatin is transferred in the ovocyte, and the form that has strengthened donorcells nuclear is reinvented, and eliminates detected defective in NT embryo's protokaryon.

Chromatin shifts produces healthy clone

SLOT makes clone embryos grow mature (accompanying drawing 51A and 51B).After being transplanted to blastaea in the recipient female, SLOT embryo's 40 days pregnancy rate is higher than NT embryo (P=0.02 significantly; Fisher ' s Test), and this trend last till and grow that mature (respectively, going out to calve is 12/59 and 23/211; P=0.05) (accompanying drawing 51A).In addition, the SLOT raising is cloned in the survival rate of birth after 24 hours and (is respectively 10/59 and 17/211; P=0.04) (accompanying drawing 51A and 51B).

By to 5 minutes (extremely unusual) animal and placenta being marked, estimate the NT (embodiment 10) of birth and SLOT clone's healthy state with 1 minute (normally).The placenta score value comprises parameter, for example placental edema, cotyledon number, size and form, amniotic fluid color, uterus shape and umbilical cord.That the animal score value comprises is functional evaluation and breathing, cardiovascular, digestion, urinary tract, muscle, bone and neural general performance.The data of each calf are shown among the accompanying drawing 51C.Box map analysis shows, derives from the animal of NT and the score value of placenta and disperses (accompanying drawing 51E) more than the animal that is produced by SLOT and those score values of placenta.SLOT and NT clone's mean birth wt does not have the significance difference; But the ratio that birth surpasses the SLOT calf of 45kg is lower than NT animal (P=0.02; Fisher) (accompanying drawing 51D and 51E).In a word, these results show that chromatin shifts has produced the offspring who lives, and has shown the evidence that developmental potency and viability improve.

Compare with NT (embodiment 10), although the SLOT offspring's who produces limited amount (n=8), the pregnancy rate when SLOT improves 40 days significantly (P=0.02) and the survival rate (P=0.04) of birth after 24 hours.The improved trend of healthy state that in box map analysis, has also reflected the animal that produces by SLOT.In addition, the situation of the big fetus (surpassing 45kg) that is produced by SLOT is less, and this has actual and economic using value in the animal operation control.

In the NT embryo, identified a plurality of nucleus defectives, comprised that Lamin A/C assembling, the rising of protokaryon TBP content and DNA raise to the resistance of DNA enzyme I.These defectives may be because fibroblastic nuclear not exclusively reinvent or by regulation and control produce to the mistake of noble cells special (for example, nuclear lamina protein A) expression of gene.Cell in vitro nuclear is reinvented and spissated chromatin is transferred to and eliminated these defectives in the ovocyte.

Reinvent nucleus by SLOT and improved the susceptibility of DNA, and may promote the chromatinic formation of transcriptional activity (perhaps lateral reactivity) DNA enzyme I.These the possibility of result help growing the expression of important function of gene conversely.For example with placenta growth, later stage pregnancy maintenance and the gene relevant with postnatal survival.SLOT also causes the inhibition to the nuclear lamina protein A genetic expression in the clone embryos.External and the in-vivo procedures that the pair cell nuclear lamina is formed is verified, can not assemble a correct cover lamin and will cause apoptosis (Steen etc., J.Cell Biol.153:621-626,2001).And, because lamin and DNA, chromatin and the mechanism of transcribing interact, in clone's embryo, it may be crucial (Gruenbaum etc. that correct nuclear lamina is rebuild for normal nucleus function, J.Struct.Biol.129:313-323,2000 and Cohen etc., Trends Biochem.Sci.26:41-47,2001).

During mitotic division or concentrate relevantly at external chromatin, for example be Chromatin Remodeling enzyme, transcription factor (for example TBP) or other potential inhibition somatocyte compositions in conjunction with the composition of DNA with release in conjunction with the composition of DNA.Removing TBP from the donor somatochrome may be with inhibition or the downward modulation that promotes among the SLOT embryos the special gene of somatocyte, and this may reduce developmental potency.From donorcells nuclear, remove the factor and mean, will make things convenient for maternal composition to be loaded on the chromatin, reinvent subsequently in the physiological protokaryon.

In a word, may in cell extract, directly reinvent somatic cell nuclear, and produce the offspring who lives.Further research for optional pair cell nuclear programming mechanism again, being used to clone or change the manipulation in vitro that breaks up purpose pair cell nuclear is a kind of useful instrument (Landsverk etc., EMBO Rep.3:384-389,2002 and Hkelien etc., Nat.Biotechnol.20:460-466,2002).The chromatin transfer has shown to strengthen grows evidence mature and raising clone viability.May further improve the efficient of cloning of mammalian animal to the operation bidirectional of this system.

Embodiment 17: adopt streptolysin O to shift (SLOT) and carry out other evidences that nucleus is more completely programmed again

When the cell of being changed processing thoroughly when donor before heredity is shifted is not sealed again, the result that the SLOT method described in the embodiment 12 is improved.In addition, use the donorcells of G1 phase to substitute to converge cell may strengthen the embryo's of the ovocyte of reconstruct and generation viability.These tests will be further described hereinafter.

The preparation of damping fluid

By 1ml water is placed Eppendorf tube, add 154mg refrigerative DTT, by the rotation thorough mixing, prepare 1 mole DTT solution.Solution is distributed in 10 1 volumes, freezing down at-20 ℃.The aliquots containig freezing several that can be thawed.The cytolysis damping fluid that is used to prepare the 100ml volume that is used for nucleus assembling/decomposition analysis contains NaCl (50mM, 1ml5M mother liquor), MgCl ₂(5mM, 0.5ml1M mother liquor), Hepes, and pH8.2 (20mM, the 2ml1M mother liquor, pH8.2) and water (96.5ml).Five equilibrium damping fluid and storage under 4 ℃.When the preparation lysate, pH descends about 1.For the preparation of mitotic division extract, add 10mM EGTA (1ml1M mother liquor), add 95.5ml water, rather than 96.5ml.Before the use, add following material: DTT (1 μ l/ml solution, be kept at the 1M mother liquor under-20 ℃, the 1mM ultimate density), PMSF (10 μ l/ml solution 100mM mother liquors, the 1mM ultimate density), the CAL mixture (contains 10 μ l CAL mixtures in every ml soln down at-20 ℃, be that ultimate density is that each of 10 μ g/ml presses down the curdled milk proteinase inhibitor, press down the enzyme peptide and press down bright enzyme peptide), Pepstatin A (10 μ l mother liquors in 20 ℃ of following every ml solns, the ultimate density of 10 μ g/ml) and cytochalasin D (from 1 μ l/ml of the 1mg/ml mother liquor under-20 ℃, the ultimate density of 1 μ g/ml).For Luo Keda azoles 1000X mother liquor solution, in DMSO, prepare the Luo Keda azoles of 1mg/ml, and store with the aliquots containig of 160 μ l.

In order to prepare streptolysin O mother liquor (SLO), with one bottle of SLO (Sigma S-5265; 25,000 units are stored under 4 ℃ with powder type) be dissolved in the 400 μ l water thorough mixing.All the elements thing is transferred in the 1.5ml conical tube, be divided into the aliquots containig of 10 μ l, be chilled under-20 ℃.Mother liquid concentration is " 1OX ".In order to prepare protein enzyme solution, 3ml TLHepes and 9mg proteolytic enzyme (SigmaP-8811) are added in the 1.5ml conical tube, mix by rotation.By 0.22 μ m syringe filter with this solution direct filtration in TL Hepes.Because cell doubles volume, ultimate density is 1.5mg/ml.For HECMHepes, made up NaCl (114mM, 6.662g), KCl (3.2mM, 0.239g), CaCl ₂2H ₂O (2.0mM, 0.294g), MgCl ₂6H ₂O (0.5mM, 0.102g), Pen/Strep (10ml, Sigma P3539 Pen/Strep, the ultimate density of 100U/ml and 100 μ g/ml), phenol red (5 μ g/ml, 1ml) and water (being enough to cumulative volume is increased to the content of 990ml).Then, add 100X A.A. (10ml), and Sodium.alpha.-hydroxypropionate (10mM, 1.44ml), Sodium.alpha.-ketopropionate (0.1mM, 0.011g), NaHCO ₃(2mM, 0.168g) and HEPES (10mM, 2.38g).Final solution has the infiltration volumetric molar concentration of 260-270mOsM.Add 3g bovine serum albumin (cut V) then, and be 7.4 pH regulator.Come filtering solution by 0.22 μ M filter, and be stored under 4 ℃.

For ATP mother liquor solution (Sigma A3377:100mM mother liquor, preparation 100x), combination water (1ml) and ATP (0.055g), and-20 ℃ of following freezing 10 μ l aliquots containigs.For phosphocreatine (Sigma P7936:1M mother liquor, preparation 100x), combination water (1ml) and phosphocreatine (0.255g), and-20 ℃ of following freezing 10 μ l aliquots containigs.(Sigma C3755:2.5mg/ml mother liquor, 100x), combination water (1ml) and sarcosine kinase (0.0025g) are-20 ℃ of following freezing 10 μ l aliquots containigs in order to prepare sarcosine kinase.For the preparation of ATP generation body, water prepares 100mM ATP mother liquor, 1M phosphocreatine and the 2.5mg/ml sarcosine kinase mother liquor (100x) of equal proportion, mixes, and stores as frozen soln.The ATP generation structure is remained on ice up to use.Add ATP generation structure (1.2 μ l) in the extract (40 μ l), rotation mixes this solution.The ultimate density of ATP generation structure in extract is 1mM ATP, 10mM phosphocreatine and 25 μ g/ml sarcosine kinases.

The preparation of mitotic division extract

1,000,000 cells of one bottle of 1.5-2 thaw.Cell is assigned in two T75 flasks, grown two days or converge up to them.Cell goes down to posterity in 6-8 T75 flask, grows into and converges.Cell is by trypsinized, and with the hemocytometer counting, 300 ten thousand cells joined in each T150 flask, and T150 flask as much as possible is used with obtainable cell quantity.With standard method (for example, Collas etc., J.Cell Biol.147:1167-1180,1999 reach reference wherein), with 0.5-1 μ g/ml Luo Keda azoles synchronization cell system (for example, inoblast for example former generation inoblast, epithelial cell or unlimited reproduction and anosis cell be the MDBK cell for example) 17-20 hour, 70-80% merges in m period.By shaking off of m period, the cell that results are synchronized.Shake vigorously with heavy-handed multiple a beating that each contains the flask of cell.Mitotic cell breaks away from and swims in the nutrient solution.Under 4 ℃, with the cell gathered in the crops in the conical tube of 50ml centrifugal 10 minutes with 500xg.Abandoning supernatant is resuspended in cell precipitation in the physiological saline of the phosphoric acid buffer of cold first Ca/Mg of 50ml altogether (PBS).If desired, a plurality of cell precipitations can be pooled in the test tube of a 50ml.Under 4 ℃,, and repeat above-mentioned washing step with 500xg eccentric cell 10 minutes.Determine the volume of cell precipitation, cell precipitation is resuspended in the ice-cold cytolysis damping fluid that contains proteinase inhibitor (being DTT and PMSF) of about 20 volumes.

Then, under 4 ℃, came sedimentation cell in centrifugal 5 minutes with 500xg.Abandoning supernatant is determined the volume of cell precipitation.Cell precipitation is resuspended in the cytolysis damping fluid that contains all proteinase inhibitor that is no more than 1 volume.Cell was cultivated on ice 1 hour, made cell expansion.Use most advanced and sophisticated ultrasonoscope, pair cell suspension carries out ultrasonication, up to all cell ruptures.Under phase microscope, monitor cytolysis.Desirably, before carrying out next procedure, 90% cell is dissolved.Ultrasonication can be lengthened to required length; Ultrasonic treatment time will change along with being used to prepare the cell type of extract.Alternately, carry out Dounce homogenate with glass mortar and pestle (refiner) and come dissolved cell, desirably, dissolved up at least 90% cell.Cell lysates is placed the 1.5ml centrifuge tube, and under 4 ℃, use tabletop refrigerated centrifuge with-15, centrifugal 15 minutes of 000xg.Place in the cold chamber whizzer or refrigerator, carry out balance.From refrigerator, take out test tube, and be placed on ice immediately.Carefully collect supernatant liquor with 200 μ l transfer pipet tips.This supernatant liquor is a mitotic division kytoplasm extract.Extract is placed on ice another test tube.The extract that to collect from a plurality of test tubes pools together.

There are two kinds of alternative selections in this stage.Cell extract is distributed on ice the test tube every pipe 41 μ l.Rapid freezing extract in liquid nitrogen immediately, and be stored in-80 ℃ the frigorimeter up to use.This from 15, the extract of the centrifugal preparation of 000xg is known as MS15 (perhaps mitotic division kytoplasm extract).Alternately, the MS15 extract is placed ultracentrifugation test tube on ice (for example be suitable for SW55 Ti rotor; Beckman).Test tube is crushed when preventing ultracentrifugation if desired, then covers test tube up to the top with mineral oil.Under 4 ℃, with 200, the centrifugal extract of 000xg 3 hours is with the membrane vesicle that contains among the precipitation MS15.In centrifugal latter stage, discard mineral oil.Collect supernatant liquor carefully, and place cold 1.5ml test tube on ice.If desired, use multiple supernatant liquor.This supernatant liquor is known as MS200 (the perhaps extract of mitotic division kytoplasm).Five equilibrium MS200 extract, and carry out described freezing as being used for the MS15 extract.

Chromatin shifts

In the day before yesterday of clone operations, prepare one and contain 1,000,000 fibroblastic 35mmNunc or 6 T75 flasks that contain 500,000 cells, be used to shake off.In the morning of clone operations, the α MEM perfect medium that contains 15% irradiated FBS in all flasks is changed, to remove cell debris and dead cell.Before carrying out cell permeabilization processing and the reaction of mitotic division extract, by serial dilution SLO mother liquor (for example, 1X, 0.5X, 0.3X and 0.1X dilution), the minimum SLO concentration that being identified for 80-90% cell is required was cultivated 30 minutes in about 38.5 ℃ water-bath, dyeed with iodate third ingot then.

With trypsinase or cellular segregation damping fluid cell is separated from converge culture or new cells transfected culture, be placed in the 15ml conical tube, use the HankShi balanced salt solution (HBSS) of no Ca/Mg to pass through centrifuge washing 1 time.Cell is resuspended among the 1mlHBSS, and pair cell is counted to determine concentration.At room temperature, with about 50,000-100,000 cell suspension 100 μ l HBSS (Gibco BRL, cat.No.14170-120) in.Add the previous 5 μ l SLO mother liquors of determining concentration.Culturing mixt is 30 minutes in about 38.5 ℃ of water-baths.In culturing process, pat test tube 2-3 time gently, still be in suspended state to guarantee cell.The room temperature PBS (no Ca/Mg) that adds 200 μ l volumes is by blowing and beating thorough mixing gently.At room temperature, in desk centrifuge, with 500xg eccentric cell 5 minutes.Discard whole supernatant liquors.Precipitate very for a short time, can not clearly be seen.

The mitotic division extract that contains the ATP generation structure that adds 40 μ l volumes, this solution of thorough mixing.In above-mentioned 30 minutes culturing process, the preparation extract.The one bottle of 40 μ l extract that thaw adds in the ATP generation structure of 1.2 μ l.Thorough mixing solution is placed at room temperature.Culturing mixt is 30 minutes in 38.5 ℃ of water-baths, pats test tube once in a while gently.The room temperature perfect medium (α MEM+15% foetal calf serum) that adds 500 μ l volumes, in the incubator that is stored in 38.5 ℃ of uncapping up to use.At room temperature, in desk centrifuge with 500xg eccentric cell 5 minutes.Cell precipitation is resuspended among the 1ml room temperature TL Hepes, and transfers in the 15ml conical tube.The 3mg/ml proteolytic enzyme (filtrations) that is dissolved in TL Hepes that adds the 1ml volume, feasible final protease concentration with respect to cell is 1.5mg/ml, 30 seconds of culturing mixt.Add TLHepes to be full of the 15ml conical tube, cover tight test tube and centrifugal 5 minutes with 2300rpm.From cell, remove TLHepes, and 150 μ l TL Hepes are joined in the cell.Clone mark test tube according to suitable is placed on the warm table.

During the preparation cell, adopting suitably, the Transplanted cells suction pipe of size prepares the operator's console that is used for Transplanted cells.About 50 non-nucleus egg mother cells are placed the TL Hepes drop of 50 μ l under the black mineral oil of 100mm culture dish.Cell after the washing is placed the drop with non-nucleus egg mother cell.Abundant cell is used for heredity transfers to all ovocytes.Avoid carefully using too much cell, make them spatially block operational tool.A non-nucleus egg mother cell is placed on the fixer, a cell is placed ovum week crack under the zona pellucida, make the plasma membrane of this cells contacting ovocyte.For the ease of merging, cell is placed on approaches polar body or relative with polar body.Contain the cell that shifts at all non-nucleus egg mother cells after, from droplet, take out ovocyte, be placed among the HECMHepes of 35mm culture dish of the preheating that is used for merging.

The result

The result of donorcells in the result of donorcells and the table 11 in the table 12 relatively, the cytolemma of the donorcells in the table 12 was not sealed before heredity is shifted again, and the cytolemma of the donorcells in the table 11 was sealed before heredity is shifted again, the result shows, no longer seals the cytolemma of being changed processing thoroughly and can improve cloning efficiency.In addition, table 12 uses G ₁The donorcells of phase substitutes and to converge the viability that cell can improve the embryo of the ovocyte of reconstruct and generation.Data in the table 11 use the fibroblastic extract from Niu Yuandai inoblast and donor ox fetus to obtain.The MDBK cell also successfully is used for producing the extract of programming again.

Table 11: use the fetal development of HAC clone: have the NT that seals donorcells again and compare with SLOT

Table 12: use the embryo's of Δ HAC and Δ Δ HAC clone growth: do not have seal donorcells again converge donorcells (CTC) and G ₁Phase donorcells (CTD) is compared

Handle	Sum	Blastaea (%)	Acceptor	Gestation 40d (%)	Gestation 60d (%)
Handle	Sum	Blastaea (%)	Acceptor	Gestation 40d (%)	Gestation 60d (%)	ΔHAC CTC	1729	185(15)	68	14/39(36)	5/13(38)
ΔHAC CTD	1177	181(22)	68	25/44(56)	5/22(33)	ΔHAC CTC	1729	185(15)	68	14/39(36)	5/13(38)
ΔHAC CTD	1177	181(22)	68	25/44(56)	5/22(33)	ΔΔHAC CTC	1230	147(17)	60
ΔΔHAC CTD	521	95(26)	29			ΔΔHAC CTC	1230	147(17)	60
ΔΔHAC CTD	521	95(26)	29			Amount to	4657	608(19)	225

Embodiment 18: be used to produce chimeric mammiferous method

It is generally acknowledged that many spontaneous abortions that come to take place in the process of cloning mammal in the employing traditional method are owing to the placenta deformity produces, rather than the problem of fetus.Therefore, with mainly being developed from the placenta tissue of a source (for example in vitro fertilization, natural or parthenogenesis activated embryo) and the method for mainly producing chimeric embryo from the fetal tissue of another source the nuclear transfer embryo of heteroantibody (for example, coding).Have mainly the chimeric embryo that activates the placenta tissue that embryo's cell obtains from vitro fertilization, natural or parthenogenesis and more be similar to natural placenta tissue, and produce the offsprings of surviving more.Preferably, the major part of progeny cell derives from the cell from nuclear transfer embryo, thereby has in fact and the identical genome of donorcells that is used to produce nuclear transfer embryo.

In a kind of this method, be injected into the fine and close embryo's of coding heteroantibody periphery (for example between zona pellucida and embryo itself) from embryo's in vitro fertilization cell, this embryo is with any production the in traditional core implantation method or other cloning process described here.In a kind of alternative method, under discerning with the condition of producing single chimeric embryo mutually from each embryo's cell, to cultivate with the fine and close embryo's who comes the own coding heteroantibody cell from the cell of the embryo in vitro fertilization before fine and close, this densification embryo (for example produces with one of cloning process of the present invention, the cell that adopts the chromatin of programming again or changed processing thoroughly is as donor source) (Wells and Powell, Cloning 2:9-22,2000).In these two kinds of methods, preferably be incorporated in the placenta from embryo's in vitro fertilization cell, and preferably be incorporated in the fetal tissue from the cell of nuclear transplantation method.These methods will be further described hereinafter.Use the nuclear transfer embryo that does not contain the heteroantibody gene, obtain these results; But, contain the nuclear transfer embryo heteroantibody gene and/or that in prion gene, contain sudden change and also be expected to obtain similar result.

The fibroblastic separation of G1 phase

For fibroblastic separation of G1 phase of producing nuclear transfer embryo as donor, use previously described " shaking off " method (Kasinathan etc., Nature biotech.19:1176-1178,2001).In brief, before separation 24 hours are with 5.0 x 10 ⁵Individual cell places and contains in the 100mm tissue culturing plate of α-MEM that 10ml added FCS.Second day, wash this plate with PBS, the 1-2 before separation hour replacing substratum.Go up the dull and stereotyped 30-60 of vibration second at Vortex-Genie 2 (Fisher Scientific, Houston, TX, middling speed) then.Remove substratum, with 500x g centrifugal 5 minutes, precipitation is resuspended in 250 μ l interpolation among the MEM of FCS.This cell suspending liquid by the new splitted cell doublet that connects by cytoplasmic bridge, part is unicellular and the cell in mid-term or later stage is formed.The donorcells that is used as nuclear transplantation by the cell doublet of cytoplasmic bridge connection.

Nuclear transplantation, activation and embryo culture

Basically according to previous description, carry out nuclear transplantation (Cibelli etc., Nature Biotech.16:642-646,1998 with isolating G1 phase inoblast; Kasinathan etc., Biol.Reprod.64:1487-1493,2000).After maturation about 18-20 hour, with the ovocyte stoning of maturation in vitro, (Hoechst 33342, and Sigma) mark confirms that karyomit(e) is removed by two benzimides under UV light.Adopt single 2.4kV/cm burst process 20 microseconds (Electrocellmanipulator 200, Genetronics, San Diego CA), merges these kytoplasms-donorcells doublet.As discussed previously, in maturation back 30 hours, (Cal Biochem, San Diego CA) activated 4 minutes and with being dissolved in ACM substratum (100mM NaCl, 3mM KC1,0.27Mm CaCl with Calcium ionophore (5 μ M) with the ovocyte of reconstruct and contrast ₂, 25mM NaHCO ₃, 1mM Sodium.alpha.-hydroxypropionate, 0.4mM pyruvic acid, 1mML-glutaminate, 3mg/ml BSA (not fatty acids) 1%BME amino acid and 1%MEM non-essential amino acid; All available from Sigma) in 10 μ g cycloheximides and 2.5 μ g cytochalasin D (Sigma) activate 6 hours (Liu etc., Mol.Reprod.Dev.49:298-307,1998; Presicce etc., Mol.Reprod.Dev.38:380-385,1994).After the activation, at HEPES buffering hamster embryo culture medium (HECM-HEPES, 114mM NaCl, 3.2mM KCl, 2mM CaCl ₂, 10mM Sodium.alpha.-hydroxypropionate, 0.1mM Sodium.alpha.-ketopropionate, 2mM NaHCO ₃, 10mMHEPES and 1% BME amino acid; Sigma) the washing ovum is 5 times in, and places the 4-hole tissue culturing plate of containing mouse fetal inoblast and 0.5ml embryo culture medium to cultivate, and covers the mineral oil (Sigma) of 0.2ml through embryo testing on the substratum.Place 25-50 piece of embryo in each hole, under 38.5 ℃, at 5%CO ₂Atmosphere surrounding in cultivate.At the 4th day, in substratum, add 10%FCS.At the 7th and 8 day, the developmental state in blastaea stage grown in record.

Ox is in vitro fertilization

Produce as being used to of describing early and to implement (Collas etc., Mol.Reprod.Dev.34:224-231,1993) in vitro fertilization the ox embryo in vitro fertilization.Ooze Percoll gradient (Parrish etc., Theriogenology 24:537-549,1985) with seminal fluid TL mother liquor preparation 45% and 90% etc.To be added to the top layer of gradient from the freeze-thaw ox seminal fluid of a bull, with centrifugal 30 minutes of 700xg (2000rpm uses 6.37 inches termination radius).Determine the sperm concentration in the precipitation, the dilution sperm makes that the ultimate density of the time of fertilization is 10 in seminal fluid TL (seminal fluid TL mother liquor, 1mM pyruvate salt, 6mg/ml BSA and 1%PS) ⁶Sperm/ml.Ripe back 22 hours; the washing ovocyte is 3 times in TL HEPES; place the 480 μ l fertilization TL (Bavister etc. in Nunc hole; Biol.Reprod.28:235-247; 1983) in; wherein contain 6mg/ml BSA, 0.2mM pyruvate salt, 20 μ M Trolovols, 10 μ M hypotaurine, 1mM suprarenin (Leibfried etc., J.Reprod.Fertil.66:87-93,1982) and 0.004 μ g/ml heparin.Add 20 μ l seminal fluid to produce 10 ⁶Sperm/ml is to the ultimate density of 50 ovocytes.Culture condition is identical with the above-mentioned condition that is used for nuclear transplantation.The rate of fertilization that gets according to former caryogenesis surpasses 90%.

Chimeric nuclear transfer embryo

At after fertilization about 96 hours, before densification, the embryo in vitro fertilization (6-12 blastomere) of results 8-cell stage.Remove zona pellucida with proteolytic enzyme (3mg/ml is dissolved in TL-HEPES).Carefully monitor the dissolving of zona pellucida with dissecting microscope.When zona pellucida begins dissolving to occur (about 2 minutes), the taking-up embryo also washs in TL-HEPES, transfers in the 30mm culture dish that contains the HankShi balanced solution, cultivates 30 minutes down at 37.5 ℃.Embryos' blastomere is transferred in the TLHEPES droplet (50 μ l) under the mineral oil in the 100mm culture dish before will be from these fine and close.At the 4th day, select the nuclear transfer embryo of 8-16 cell stage, transfer in the same droplet that contains blastomere.These nuclear transfer embryos comprise fine and close preceding embryo (for example, the embryo of 8 cell stages) and fine and close embryo (for example, the embryo of 16 cell stages).Then, adopt the micromanipulative technique of standard, 4-6 blastomere transferred in the nuclear transfer embryo with inclined-plane microtubule (35 μ m diameter).After shifting blastomere, as be used for the method for nuclear transfer embryo and cultivate the embryo described.

At the 7th and 8 day, chimeric embryo is estimated to the growth in blastaea stage.Also analyze whether there is cytolemma dyestuff DiI in the blastaea, before the cell from embryo in vitro fertilization is injected into nuclear transfer embryo, in this cell, add dyestuff DiI.At the 4th day labeled cell, and observed at the 7th day.This dyestuff is held in the offspring's of the cell that is colored at first a few cell differentiation, makes and can break up the post analysis chimeric embryo in a few cell.Analyze based on this, be incorporated in the chimeric embryo from embryo's in vitro fertilization cell.If desired, can adopt standard method, with the probe of nucleic acid that is specific to embryo in vitro fertilization or nuclear transfer embryo carry out fluorescence in situ hybridization (FISH) (referring to, for example, Ausubel etc., Current Protocolsin Molecular Biology, John Wiley ﹠amp; Sons, New York, 14.7.1-14.7.12,1995).The cell that this fish analysis can be used to determine to derive from various embryos at the chimeric embryo of vitro culture (for example, determine that how many ratio cells are incorporated in the inner cell mass and the cell of how many ratios is incorporated in the trophoderm), fetus or by the distribution situation among the offspring of this Embryo Production.Alternately, reporter gene such as green fluorescent protein can be added in the cell from one of embryo, and the cell that is used for monitoring chimeric embryo is attached to the situation of placenta and various fetal tissues.

Embryo transfer

At the 7th and 8 day, be transplanted in the receptor cow that the 6th and 7 day synchronization handles deriving from nuclear transfer embryo and chimeric nuclear transfer embryo 1 grade and 2 grades of nuclear transplantation blastaeas.Adopt single injection Lutalyse (Parmacia ﹠amp; Upjohn, Kalamazoo MI) comes synchronization to handle acceptor, the observation of then oestrusing.After embryo transfer the 30th and 60 day, check the existence of conceptus by ultrasonic scanning, carried out examination per rectum in after this per 30 days, up to the 240th day.In table 13, compared chimeric embryo and by merging contrast embryo that transgenic cattle inoblast and ovocyte produce the 40th day pregnant situation.These results show that more chimeric embryo was survived by the 40th day.

Table 13: embryo transfer and pregnant situation

Be used to produce the alternative of chimeric embryo

Can adopt standard method to improve the above-mentioned method that is used to produce chimeric embryo.For example, natural embryo is obtained in operation from Mammals (for example ox), perhaps with standard technique the ovocyte parthenogenesis is activated, and uses this ovocyte to substitute embryo in vitro fertilization.If desired, the less cell from vitro fertilization, natural or parthenogenesis activated embryo (for example 1,2,3,4 or 5 cell) is expelled in the nuclear transfer embryo, reduce the ratio of the cell of injecting, the offspring of these cells is incorporated in the fetal tissue.Alternately, can inject, improve by the injection cell and be incorporated into the ratio of the cell offspring in the placenta tissue than many cells (for example 6,7,8,9,10,11 or more a plurality of cell).In addition, can use the cell that is in other cell stages from the embryo.For example in vitro fertilization, the natural or parthenogenesis activated embryo who is in 4 cells, 8 cells, 16 cells, 32 cells, 64 cells, 128 cells, 256 cells, 512 cell stages or later stage cell stage can be expelled in the nuclear transfer embryo that is in 4 cells, 8 cells, 16 cells, 32 cells, 64 cells, 128 cells, 256 cells, 512 cell stages or later stage cell stage.Injected cell can be in identical cell stage or be in different cell stages with nuclear transfer embryo.In one embodiment, in vitro fertilization, natural or parthenogenesis activated embryo has the ploidy higher than nuclear transfer embryo (for example, dna content is 4n), and this further makes injected cell be partial to trophoderm (promptly, embryo's outermost layer cell mainly forms placenta tissue).If desired, kept all or part of zona pellucida by the injection cell peripheral, rather than before injection, be removed.

In other alternative, under allowing to discern with the condition of producing single chimeric embryo mutually from various embryos' cell, will be from vitro fertilization, natural or parthenogenesis activated embryo's before fine and close or fine and close cell with cultivating (Wells and Powell from the cell of the nuclear transfer embryo before fine and close, Cloning 2:9-22,2000).Be considered to mainly form trophoderm from cell in vitro fertilization, natural or gynecogenic embryo, finally form placenta tissue, and be considered to mainly form inner cell mass, finally form fetal tissue from the cell of nuclear transfer embryo.Cell from two kinds of embryos can be in the same cell stage or be in different cell stages.The cell from various embryos of identical or different quantity combines to form and assembles embryo (aggregation embryo).

If desired, the clone fetus of self-generating in the future or clone offspring's cell is used for second and takes turns nuclear transplantation, with the outer clone's of delivery capacity offspring.Also can be frozen from the cell of cloning fetus or clone offspring first, form clone as the donorcells source of the outer ungulate of cloning of delivery capacity.

Embodiment 19: to the detection of people Ig expression

Can be after birth immediately the calf of the nucleic acid that kept HAC or other coding heteroantibodies be detected, comprise evaluations (1) xenogenesis Ig expression, (2) to the reaction of immunization, (3) affinity maturation and the transmission of (4) HAC in the offspring.

Can be by from animal, taking a blood sample, by ELISA, RT-PCR or facs analysis (referring to, the open WO01/35735 of PCT for example), detect the expression that whether has people's heavy chain and light chain, the expression that comes monitor Ig.Generate people Ig in case determine animal, with the tetanus toxin immunization animal that is dissolved in the adjuvant.After immunization, animal is carried out weekly blood sampling, and determine antigenic reaction by ELISA or FACS, compare with the previous blood sample of collecting before the immunization.Inoculate back 1 month in first immunisation, animal is carried out booster immunization with the antigen of aqueous solution form.。1 week behind booster immunization, from the animal blood sampling, and definite by ELISA or FACS to antigenic reaction, with previous blood sample comparison.The feasible heavy chain isotype that can measure most reaction titre and generation of ELISA or facs analysis.These data make can be determined the rising of antibody titers and whether the kind conversion takes place.Estimated also average affinity determines whether the affinity maturation takes place in to antigen reactive process.

After obtaining transgenic cattle as mentioned above, these oxen are used to production transgenosis Ig, preferably are people's Ig, also may be the Ig of other species, for example dog, cat, non-human primates, other ungulates, for example sheep, pig, goat, Muridae such as mouse, rat, cavy, rabbit etc.As noted, known between species, the Ig gene is guarded.

The transgenosis antiserum(antisera) and the milk that contain heteroantibody

Ox (or other ungulates) produces antiserum(antisera), the antigen that any antigen that this antiserum(antisera) opposing endogenous exposes or opposing external source are used.For example, antigen is administered to ungulate, to generate and this antigen reactive expectation antibody, antigen comprises as pathogenic agent (for example bacterium, virus, protozoon, yeast or fungi), tumour antigen, acceptor, enzyme, cytokine etc.The exemplary pathogenic agent that is used for antibody producing comprises, but be not limited to, hepatitis virus (for example, hepatitis C virus), immunodeficiency virus (for example HIV), simplexvirus, parvovirus, enterovirus, Ebola virus, rabies virus, Measles virus, vaccinia virus, suis (for example streptococcus pneumoniae), influenzae (hemophilus influenzae), neisserial (Neisseria meningitidis), corynebacterium diphtheriae, influenzae (Hemophilus pertussis), clostridium (Clostridium botulinum), staphylococcus, pseudomonas (for example Pseudomonas aeruginosa), respiratory syncytial virus (RSV).

One or more pathogenic agent can be administered to genetically modified ungulate, can be used for preventing, stablizing or treat the hyper-immuneserum of specified disease with production.For example, the pathogenic agent that the respiratory tract infection of suffering from children is relevant is applied to transgenic ungulates, to produce the antiserum(antisera) (for example, streptococcus pneumoniae, hemophilus influenzae and/or Neisseria meningitidis) with these pathogenic agent reactions.Selectively, before being administered to ungulate, handle these pathogenic agent, with the toxicity that reduces them (for example, by heating or be exposed to chemical reagent such as formaldehyde).

In order to produce wide spectrum Ig, various pathogenic agent (for example, pathogenic agent various bacteria and/or virus) are administered to transgenic ungulates.This hyperimmunization antiserum(antisera) is used to the transmissible disease in prevention, the stable or treatment Mammals (for example people), can be used for the treatment of the Mammals that suffers from heredity or acquired immune deficiency especially.

In addition, the antibody of producing by method of the present invention can be used to suppress immunity system, for example treats neuropathy, and eliminates specific human cell and regulate specific molecular.For example, anti-atopic antibody (antibody that promptly suppresses other antibody) and can be used to treat autoimmune disease or neuropathy (for example, the neuropathy that causes by inflammation) with the antibody of T cell, B cell or cytokine reaction.These antibody never obtain in the transgenic ungulates of administration of antigens, perhaps obtain from be given the antigenic transgenic ungulates as B cell, T cell or cytokine (for example TNF α).

Transgenosis antiserum(antisera) by the not transgenic ungulates production of administration of antigens can be used to produce medicine, and this medicine comprises people's polyclonal antibody, preferred human IgG molecule.These human antibodies can be used to substitute from human isolated antibody, as intravenously immunoglobulin (Ig) (IVIG) therapeutical agent.Randomly, in can enrichment transgenosis antiserum(antisera) with one or more interested antigen reactive antibody.For example, can use standard technique, as (Current Protocols in Molecular Biology, the 2nd volume, 11.13.1-11.13.3, John Wiley ﹠amp such as Ausubel; Sons, 1995) those methods described in are come the purifying antiserum(antisera).Preferred purification process comprise with the pearl of antigen or antibody sandwich precipitate, column chromatography such as affinity chromatography, magnetic bead protein affinity purification, and carry out elutriation with combining antigenic flat board.In addition, the transgenosis antiserum(antisera) contacts with one or more interested antigens, according to the increase of antibody/antigen mixture size the antibody of conjugated antigen and not combined antibody is separated.Can also come the IgG purification molecule with albumin A and/or Protein G.If the expression of endogenous antibody is not removed, can people's antibody of expectation antibody or ungulate/people's chimeric antibody with the endogenous ungulate be separated with the antibody of albumin A and/or anti-people Ig light chain λ (Pharmingen).Albumin A is higher than avidity to ox Ig heavy chain to the avidity of people Ig heavy chain, and the expectation Ig molecule that can be used to contain two people's heavy chains separates with other antibody that contain one or two ungulate heavy chain.The Ig molecule that the antibody of anti-people Ig light chain λ is used to have two people Ig λ chains separates with the molecule that those have one or two ungulate Ig light chain.Additionally or alternately, one or more antibody that are specific to ungulate Ig heavy chain or light chain are used to negative screening step, contain the heavy chain of one or two ungulate and/or the Ig molecule of light chain with removal.

The antiserum(antisera) itself that obtains can be used to resist a kind of antigenic passive immunization.Alternately, this antiserum(antisera) has the purposes of diagnostic, preventative or purifying, for example is used for antigen is carried out purifying.

Alternately, after using antiserum(antisera), from transgenic cattle, separate the B cell, be used to prepare hybridoma.For example, adopt standard technique to merge hybridoma (Mocikat, J.Immunol.Methods 225:185-189,1999 that produce the interested monoclonal antibody of secretion from B cell and the myeloma cell of transgenosis ungulate; Jonak etc., Hum.AntibodiesHybridomas 3:177-185,1992; Srikumaran etc., Science 220:522,1983).Preferred hybridoma comprises that those are by the B cell hybridoma that production obtains with merging from the mammiferous myeloma cell of kind identical with transgenic ungulates or species.Other preferred hybridomas are from Balb/C mouse or people.In this case, generate a kind of hybridoma that produces the xenogenesis monoclonal antibody of anti-specific antigen.For example, this technology is used to produce the monoclonal antibody of (depending on specific artificial chromosome) such as people, cat, dogs, and this antibody is specific to pathogenic agent.The method that is used to screen the hybridoma of producing the antibody with desired characteristic is known, and these characteristics are that enhanced is in conjunction with affinity, avidity.

Alternately, from the B cell of transgenic ungulates by genetic modification, to express oncogene, for example ras, myc, abl, bcl2 or neu, perhaps with DNA or RNA viruses, for example Epstein-Barr virus or SV40 virus transfection (Kumar etc., Immunol.Lett.65:153-159,1999; Knight etc., Proc.Nat.Acad.Sci.USA 85:3130-3134,1988; Shammah etc., J.Immunol.Methods 160-19-25,1993; Gustafsson and Hinkula, Hum.Antibodies Hybridomas 5:98104,1994; Kataoka etc., Differentiation 62:201-211,1997; Chatelut etc., Scand.J.Immunol.48:659-666,1998).The B cell of the unlimited reproduction that generates can also be used to antibody endless on the production theory.Because Ig is also secreted in the milk of ungulate, ungulate milk also can be used as the source of heteroantibody.

Other embodiments

From foregoing description, can find out significantly, can carry out various changes and modifications to invention described herein, so that it is suitable for various uses and situation.For example, can adopt other incubation times or temperature (for example 25,30,35,37,38.5 or 40 ℃ or higher or lower temperature) to change processing cytolemma, programme donor genetic stocks, closing cell's film more thoroughly, and/or with transfer of genetic material in ovocyte.These embodiments are also in the scope of following claims.

All publications of mentioning in this manual all are hereby incorporated by, its degree with each independently publication or patent application clearly and respectively indicated be incorporated herein by reference the same.

Sequence table

<110>Hematech LLC

Kirin Beer Kabushiki Kaisha

＜120〉transgenic ungulates of prion protein activity reduction and uses thereof

<130>

<150>PCT/US03/35720

<151>2003-11-10

<150>US 60/506,901

<151>2003-09-26

<150>US 60/425,056

<151>2002-11-08

<160>98

<170>PatentIn version 3.3

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Claims

1. comprise the ox cell of the sudden change of non-natural generation on one or two allelotrope of endogenous Protein virus nucleic acid, the expression of functional prion protein has been eliminated in wherein said sudden change basically, and wherein said cell is not a sexual cell.

2. the cell of claim 1, wherein said sudden change is hemizygous.

3. the cell of claim 1, wherein said sudden change is homozygous.

4. the cell of claim 1, wherein said sudden change comprise positive selectable marker are inserted in the Protein virus nucleic acid.

5. the cell of claim 4, wherein said positive selectable marker is an antibiotics resistance gene.

6. the cell of claim 4, wherein said positive selectable marker is operably connected on the heterogenous promoter.

7. the cell of claim 3, wherein each allelotrope comprises identical antibiotics resistance gene.

8. the cell of claim 3, wherein each allelotrope comprises different antibiotics resistance genes.

9. the cell of claim 1, wherein said sudden change comprise the STOP codon are inserted in the Protein virus nucleic acid.

10. the cell of claim 9, wherein said STOP codon are inserted into the downstream of the initial ATC codon in the exon 3 of described Protein virus nucleic acid.

11. the cell of claim 10, wherein said STOP codon inserts with the Nucleotide within 10 of described STOP codon.

12. the cell of claim 1, wherein said sudden change are not included in the one or more Nucleotide of deletion in the Protein virus nucleic acid.

13. the cell of claim 1, wherein said sudden change are included in the one or more Nucleotide of deletion in the Protein virus nucleic acid.

14. the cell of claim 1 comprises that one or more comprises one or more transgenosiss and express a kind of mRNA or by the proteinic nucleic acid of described transgene expression.

15. the cell of claim 1 comprises that one or more comprises all or part of nucleic acid of heteroimmunity sphaeroprotein (Ig) gene, one or more xenogenesis Ig molecule can take place to reset and express in wherein said gene in the B cell.

16. the cell of claim 14 or 15, comprise one or more the coding heteroantibody nucleic acid.

17. the cell of claim 16, wherein said albumen or described heteroantibody are the antibody from another kind.

18. the cell of claim 17, wherein said heteroantibody are people's antibody.

19. the cell of claim 16, wherein said antibody is polyclonal antibody.

20. the cell of claim 16, wherein said antibody is expressed in serum and/or the milk.

21. the cell of claim 14 or 15, wherein said nucleic acid is comprised in the chromosome segment.

22. the cell of claim 21, wherein said chromosome segment are Δ HAC.

23. the cell of claim 21, wherein said chromosome segment are Δ Δ HAC.

24. the cell of claim 14 or 15, wherein said nucleic acid is independent of host chromosome and is kept.

25. the cell of claim 15, wherein said nucleic acid comprise the light chain of antibody nucleic acid fragment that is not rearranged, and wherein separate with the nucleic acid fragment of all coding J gene fragments by the nucleic acid fragment of one or more Nucleotide with all coding V gene fragments.

26. the cell of claim 15, wherein said nucleic acid comprises the heavy chain of antibody nucleic acid fragment that is not rearranged, and wherein (i) separates with the nucleic acid fragment of all encoding D gene fragments and/or (ii) separate with the nucleic acid fragment of all coding J gene fragments by the nucleic acid fragment of one or more Nucleotide with all encoding D gene fragments by the nucleic acid fragment of one or more Nucleotide with all coding V gene fragments.

27. the cell of claim 1 comprises and reduces the sudden change that endogenous antibody is expressed.

28. the cell of claim 27, wherein said sudden change has reduced the expression of functional IgM heavy chain.

29. the cell of claim 28, the expression of functional IgM heavy chain has been eliminated in wherein said sudden change basically.

30. the cell of claim 27, wherein said sudden change has reduced the expression of functional Ig light chain.

31. the cell of claim 30, the expression of functional Ig light chain has been eliminated in wherein said sudden change basically.

32. the cell of claim 27, wherein said sudden change have reduced the expression of functional IgM heavy chain and functional Ig light chain.

33. the cell of claim 32, wherein said sudden change have been eliminated the expression of functional IgM heavy chain and functional Ig light chain basically.

34. the cell of claim 1, it comprises sudden change in one or two allelotrope of the endogenous nucleic acid of coding for alpha-(1,3)-galactosyltransferase.

35. the cell of claim 1, it comprises sudden change in one or two allelotrope of the endogenous nucleic acid of coding J chain.

36. the cell of claim 1 comprises the nucleic acid of coding xenogenesis J chain.

37. the cell of claim 36, wherein said J chain are people J chains.

38. the cell of claim 14 or 15, wherein said nucleic acid is independent of host chromosome and is maintained in the ox cell.

39. the cell of claim 1, wherein said cell is a somatocyte.

40. the cell of claim 1, wherein said cell are the B cells.

41. cell and myeloma cell by claim 40 are merged the hybridoma that forms.

42. be used to produce the method that functional prion protein is expressed the transgenic cattle cell that reduces, be included in and allow to be used on one or two allelotrope of endogenous Protein virus nucleic acid, to import the sudden change that non-natural takes place, under the condition with first allelotrope generation homologous recombination of first prion gene targeting vector of the expression that reduces functional prion protein and the endogenous Protein virus nucleic acid in the described cell, first Protein virus targeting vector is imported in the ox cell, thereby in described cell, import the hemizygote sudden change.

43. the method for claim 42, also be included under the condition that homologous recombination takes place in cell between second allelotrope that allows first carrier and endogenous Protein virus nucleic acid, described first carrier is imported in the described cell, thereby in described cell, import the homozygote sudden change.

44. the method for claim 42, also be included under the condition that homologous recombination takes place in cell between second allelotrope that allows second carrier and endogenous Protein virus nucleic acid, second prion gene targeting vector that will have the antibiotics resistance gene that is different from first carrier imports in the described cell, thereby imports the homozygote sudden change in described cell.

45. the method for any one among the claim 42-44, wherein said cell are the ox inoblasts.

46. the method for claim 45, wherein said cell is a bovine fetal fibroblast.

47. nucleic acid that is used for lowering the expression of the functional prion protein of ox cell, it comprises box, this box along 5 ' comprise to 3 ' direction, have first homology zone, the positive selectable marker of the sequence substantially the same and have second homology zone with the substantially the same sequence in second zone of described Protein virus nucleic acid with first zone of the endogenous Protein virus nucleic acid of ox cell, wherein said first homology zone is longer than described second homology zone, and wherein said box can be incorporated in the endogenous Protein virus nucleic acid of described cell.