CN105018531A - Technology for producing transgenic mammal through intrauterine exogenous gene injection method - Google Patents

Abstract

A purpose of the present invention is to provide a method for producing transgenic mammal through an intrauterine exogenous gene injection method. The method is characterized in that an exogenous gene is injected into the maternal uterus within the specific period after the animal is subjected to breeding (the blastocyst formation stage), and parturition is normally performed so as to obtain the stably-genetic transgenic animal. Compared with other transgenic technologies in the prior art, the technology of the present invention has characteristics of simpleness, easy performing, low cost, and high success rate.

Description

Intrauterine injection foreign gene method produces the technology of transgene mammal

Technical field

The present invention relates to a kind of method preparing transgene mammal.

Background technology

Mammals Transgenic Technology is the new bio technology combined by genetically engineered and embryo engineering.It is the genome foreign DNA of artificial recombination being imported receptor by artificial method, or the section of DNA in excision acceptor gene group, thus make the genetic information of receptor (genotype and phenotype) that the change of artificial property occur, and this change can entail a biotechnology of offspring ^[1].

From the allophenic mice of 1974 ^[2]since birth, Mammals Transgenic Technology brings a new technological revolution to ambits such as developmental biology, medical science and livestock industry.Along with the development of molecular biology and biotechnology, Mammals Transgenic Technology achieves significant progress.By transgenic animal model ^[3,4,5], the genesis and development of molecule, cell, tissue, organ and individuality and aging are united, from the expression regulation rule of Time and place comprehensive angle research gene; By transgenic animal model, the generation of inherited disease, the rule of development can be studied, along with Gene targeting ^[6,7]the maturation of technology, transgenic technology may become a kind of important means of curing human inheritance's disease; Cultivate by transgenic technology the transgenic animal that disease resistance is strong, fast growth, the output of live-stock product and quality are high, and produce medicinal or nutrient protein by transgenic animal ^[8,9], provide allos organ etc. for the mankind, the final economic benefit improving livestock industry.

Since the chimera mouse of the eighties in 20th century is born, scholars constantly seek by simple to operate, cost is low, transgenic technology for the purpose of high-efficient transgenic.But, to the transgenic approach that Mammals carries out transgenosis production conventional be up till now: the technological methods such as microinjection, stem spermatogonium transfection, retroviral infection method, embryonic stem cell method, archeocyte (PGC) and sperm.

1. microinjection (microinjection) method

Microinjection (microinjection), utilizes micromanipulative technique, directly by the method for sperm injection to mature oocyte kytoplasm, is called intracytoplasmic sperm injection technology.In micrurgy environment, adopt this technological method, in the middle of protokaryon goal gene fragment good for external structure being expelled to the zygote in protokaryon period, synthesize along with acceptor zygote DNA or repair, exogenous origin gene integrator is in its genome.Microinjection is that the transgenosis effect of generally acknowledging at present is best, makes the working method that transgenic animal are most widely used ^[10,11].

The advantage of microinjection the different lengths of below 100kb and not containing in the middle of the DNA fragmentation injection protokaryon of prokaryotic vector, and can obtain the positive transgenic animal of foreign gene effective expression.But this technology also also exists many restraining factors, genomic dna can only increase and can not knock out or modify in the site of specifying, and the integration site of foreign DNA and copy number also uncontrollable; The costliness of microinjection cost, survival rate of embryo low, on especially large domestic animal, needs based on a large amount of embryos during preparation transgenic animal, add apparatus expensive, complicated operation, and technician's operative technique is required that high factor limits the application of this technology.

2 retroviral infection methods

1976, although Jaenich etc. utilize retroviral infection method to obtain positive mice first, foreign gene but showed as silence.Until 1998, Chan A.W. etc. ^[12]utilize ovocyte and the Cabot(2001 of slow virus Successful transfection ox) etc. ^[13]the porcine oocytes of Successful transfection maturation in vitro, and obtain transgenic pig, retroviral infection method is just extensively paid attention to by people again.

Retrovirus is diplornavirus, after infected cell under the effect of the ThermoScript II of self, is that template reverse transcription in host cell karyomit(e) becomes DNA with RNA.Its concrete grammar is nutrient solution vector virus being put into animal body early embryo ^[14], vector virus is injected segmentation cavity ^[15,16], vitro culture transfection sperm or ovocyte ^[17,18].Produce transgenic animal by retroviral vector method and there is impact simple to operate, not to be subject to Embryonic Stages, and gene expression efficiency is high; Unit point, singly copy integration, the advantages such as easy discriminatory analysis insertion point.But, during with this method infecting embryo cell, if foreign DNA is incorporated in embryonic gene group before First cleavage, can transgenic animal be obtained; But if integrated after occurring in First cleavage, can produce mosaic, the chimeric next generation may have pure lines transgenic animal, like this experimental period is just very long.And entrained foreign aid's gene fragment length is very restricted, generally can not more than 10kb ^[19].

Along with the further investigation to retroviral infection method, it is found that gene silencing phenomenon, its main mechanism is: trans-acting factor is combined by distinguished sequence in viral promotors affects the proviral expression of DNA; The provirus of integration and methylating of both sides host DNA sequence, suppress it to express ^[20].

Add the danger that vector virus gene has potential tumorigenesis and causes viremia, which limits and use retrovirus very by the widespread use of transgenic animal.

3 body-cell neucleus transplantings (somatic cell nuclear transplantation) method

Body-cell neucleus transplanting (somatic cell nuclear transplantation) method be by the donor somatic of vitro culture after foreign DNA transfection, pick out positive transgenic cell mass, more finally can obtain transgenic animal by somatic cell nuclear transfer technique and embryo transfer ^[21]method.Although with the method make the advantage of transgenic animal be in theory efficiency up to 100%.But that to be cost is high for its shortcoming, the efficiency of site-directed integration and nuclear transplantation is still very low.Obtain transgenic animal by transgenosiss such as inoblast, fetal fibroblast, cumulus cell and mammary gland cells at present.Schnieke etc. (1998) ^[22]the sheep fetal fibroblast cell by foreign gene transfection, and successfully obtain 3 Transgenic Sheep, Yangzhou University Cheng Yong in 1999 ^[23]teach problem group goes out goat by fetal fibroblast successful clone, within 2000, poplar passes through body-cell neucleus transplanting Adult Bovine have sharp ears inoblast successful clone transgenic cattle to medium, the same year, the seminar of NORTHWEST CHINA agriculture and forestry science and technology university professor Zhang Yong leader cultivated the first transgenic goat in the world of being cloned by adult sheep ear skin cell paste nuclear transplantation, China Agricultural University Dai Yunping (2003) etc. ^[24]teach (2008) etc. with Northeast Agricultural University Liu Zhonghua ^[25]also transgenic cattle and pig is obtained respectively.

4 embryonic stem cells (Embryonic stem cell, ES cell) method

Announce to be separated to obtain ES cell from Evans with Kaufman in 1981 ^[26]since, people are to the research of embryonic stem cell day by day deeply.Bradly (1984) etc. ^[27]confirm that the ES cell of vitro culture can develop into germline chimeras first, and obtain homozygote offspring.Liu Aimin (1994) etc. ^[28]have studied hprt gene to locate in mouse embryo stem cell and cause change characteristic.Chen Shengwei (1999) etc. ^[29]report and successfully obtain chimeric research by mouse ES cells germline, Military Medical Science Institute of China Zhou Jiang etc. ^[30]domestic the first Smad2 conditional gene targeting mouse is obtained in calendar year 2001.

ES cell is the multipotential cell system set up through vitro culture from the inner cell mass of body early embryo, and it has the morphological specificity similar to embryonic cell and differentiation character, keeps undifferentiated state, but can be induced to differentiate under proper condition when cultivating in vitro.Have stable caryogram due to it and be easy to trap foreign gene, therefore ES cell carries out gene knockout or the directed desirable cell inserting foreign gene.

The method is first by foreign gene transfected ES cells, cultivation filters out positive cell, positive cell is injected the segmentation cavity of receptor, produce chimeric animal, when ES cytodifferentiation is sexual cell, foreign gene entails offspring by sexual cell, namely from the s-generation, obtains transgenic animal.This method can be selected positive cell, realizes the site-directed integration of foreign DNA.Shortcoming is first on behalf of mosaic, not foreign gene-carrying in many chimeric animal sexual cell, and the efficiency namely obtaining transgenic animal is low and the cycle is long.

Although ES cell method success ratio is higher, genetic modification ability is accurate, and just can screen for the first time in the cell cultures stage, greatly improve genetically modified efficiency, but just establish ES clone mouse and the mankind at present, the stem cell line of not yet Erecting and improving on other animals, also has the method to need skilled embryo operation technician.

5 Sperm-mediated transgenosiss (sperm-mediated gene transfer, SMGT)

Brackett (1971) etc. ^[31]by the sperm of rabbit and after hatching altogether with the SV40 DNA that H3 marks, radioactive substance is detected at sperm head, with these with carry out artificial insemination with the sperm that the SV40 DNA that H3 marks was hatched altogether, and SV40 DNA all detected in the embryo when zygote and 2-cell, confirm that foreign gene both can enter spermatid, can also express at after fertilization.Regrettably this technology not to cause the concern of people at that time.Until 1989, wait Arezzo F etc. ^[32]with Lavitrano M etc. ^[33]all report after spermatid can be combined with exogenous DNA and produce transgenic progeny, this just attracts many scholars to be engaged in the research of this respect.But again due to (1989) such as Brinster R L ^[34]do repeated experiment and do not obtained positive findings, and SMGT technology is under suspicion.Decades then, researchist has done large quantifier elimination by Mammals, birds, fish and insects, confirms that sperm can adsorb and integrate foreign DNA, and carries exogenous DNA and enter in ovum.Even to this day, still arguement is existed to this technology, but utilize sperm to prepare transgenic animal as the carrier of foreign gene still very to make one notice, SMGT technology have two large advantage: a, can simple, efficient, quick, cheap, carry out transgenosis safely, its cost only has 1/10 of microinjection cost; B, do not need to carry out surgical procedure to animal.In this way the earliest by Lavitrano M etc. ^[33]mouse succeeds, so far rabbit ^[35], pig ^[36], ox ^[37], sheep ^[38]upper and the chicken Deng Mammals ^{[39 ~ 41]}and fish ^[42]on all in succession succeed.Its method is exactly the characteristic utilizing animal sperm to have the spontaneous ability in conjunction with foreign DNA, the sperm of maturation and foreign DNA are hatched altogether, then embryo transfer again after vitro culture after AI or IVF is directly carried out, sperm is made to carry foreign DNA, being integrated in the karyomit(e) of zygote with making foreign DNA during ovum fertilization, finally easily obtaining transgenic animal.Produce transgenic animal by SMGT technology be convenient to laboratory study and apply aborning.

Current application SMGT technology obtains transgenic animal and realizes mainly through sperm infection protocol (convoluted seminiferous tubule injection, intratesticular injection method etc.) two large approach in external sperm infection protocol (sperm and foreign DNA directly hatch method, liposome mediated transfection method and external electroporation introductory technique etc. altogether) and body.

5.1 external sperm infection protocols

External sperm infection protocol comprises: sperm and foreign DNA directly hatch method, liposome mediated transfection method and external electroporation introductory technique etc. altogether.

Sperm and foreign DNA directly hatch method altogether, be under certain condition direct sperm is mixed with exposed foreign DNA hatch altogether after, to still keep good vigor and be adsorbed with the sperm of foreign DNA, obtaining transgenic animal by the in vitro fertilization or method such as artificial insemination and embryo transfer.At molecular biological research initial stage and technical study initial stage in vitro fertilization, Brackett etc. (1971) ^[31]directly with after the SV40 DNA of exposed H3 mark and the sperm incubation of rabbit, after sperm head detects radioactive substance, through artificial insemination, embryo again when zygote and 2-cell all detects SV40 DNA, confirm that foreign gene can enter spermatid first, and can express in the embryo of after fertilization.1989, Arezzo F etc. ^[32]with Lavitrano M etc. ^[33]etc. further demonstrate that sperm has the ability in conjunction with foreign DNA.Lavitrano ^[43]find that in 2003 conditions such as transfection efficiency and the time of hatching, temperature etc. are relevant.Dong Huansheng etc. ^[44](2007) DIG end-labelling and immunohistochemistry technique is utilized to analyze mouse sperm and foreign DNA under incubation time is respectively 20min, 40min, 60min, 90min condition altogether, during the external efficiency in conjunction with internalization foreign DNA, find the combination prolongation in time of foreign DNA and sperm in 60min and increase, positive rate and the 60min difference of 90min sperm are little.The embryo detecting 2-4 cell stage under rear fluorescent microscope in vitro fertilization has GFP to be dispersed in strong and weak 4.7% (2/43) the uneven positive rate of distribution.Wang Xiaomei etc. ^[45](2005) sperm in Golden Hamster epididymis and plasmid NFkB-luc ⁺after mixed culture, the sperm of 21% is had to be combined with NFkB-luc through fluorescence in situ hybridization detection ⁺.Carry out in vitro fertilization with these positive sperms and ovum immediately, fluorescence in situ hybridization detection, the male pronucleus of zygote has found the existence of positive signal too.Science confirms that the foreign DNA entering sperm head is first integrated with Human Sperm Chromosome closely, by fertilization, foreign DNA is being passed to filial generation, and in this course, the sperm after transfection is the same with eupyrene sperm can be fertilized.Ren Hongyan etc. ^[46](2007) will with people her ²after the carrier enzyme of gene cuts purifying with go the pig fresh semen of seminal plasma to be mixed in proportion, 17 DEG C of standing 2h make foreign DNA enter sperm head, pass through artificial insemination.Finally the transgenic pig (4/20) that PCR detects acquisition 20% is carried out to piglet.Zhao Yongju etc. ^[47](2008) fresh essence and freeze the linear pEGFP-N1 plasmid that mark with ground height zinc respectively of essence and hatch transfection altogether, detect transfection efficiency with in-situ hybridization method respectively, PCR-Southern hybridization check integration, all detection has positive findings.And confirming that freeze-thaw can improve the efficiency of sperm transfection foreign DNA, its one of the main reasons is the integrity of plasmalemmae of sperms due to freezing for destroying, removes the inhibition of plasma membrane.

Electronegative DNA, with positively charged ion, can spontaneously wrap up by cationic-liposome surface, interacts and forms liposome-DNA complex, easy and spermatid plasma membrane fusion, thus enters cell interior.Rottmann etc. ^[48](1996) also artificial insemination after DNA plasmid after liposome and rabbit sperm being hatched altogether, obtains the transgene rabbit offspring that positive rate is 63%.Wang Xiaomei etc. ^[45](2005) by after the sperm mixed culture in the plasmid NFkB-luc+ of liposome and Golden Hamster epididymis, positive signal is had through in vitro fertilization obtaining in the zygote (17/118) of 14.4%.Xiao Hongwei etc. ^[49](2006) 1 transgenosis piglet is obtained with liposome-hCD59 gene composite by artificial insemination mode.Current liposome mediated-method have produce easy, toxicity is low, without advantages such as risk of infection.

Electroporation technology is the state and the permeability that utilize pulsed electrical field to change cytolemma, reaches DNA transfered cell and impels cell that the object merged occurs.Electroporation has the plurality of advantages such as easy and simple to handle, transfection efficiency is high than traditional calcium phosphate and liposome transfection, particularly to the cell that those other methods are difficult to prove effective, there is clear superiority, the efficiency of its transgenosis improves 1-2 the order of magnitude than chemical method usually, but its influence factor is also many, the frequency as the waveform of the height of voltage, electricimpulse, amplitude and time length, electricimpulse) and the factor such as damping fluid relevant.Gangne MB etc. ^[50]niu Jingzi is mixed with foreign gene and is placed on pulsed electrical field, find that the quantity of sperm foreign gene-carrying improves 5 ~ 8 times.Horan R etc. ^[51](1992), after the sperm with electroporation process pig, discovery sperm carries foreign aid's gene and improves 5% ~ 10% than hatching altogether, and namely electroporation can significantly improve the transfection efficiency of sperm.Rieth A etc. ^[52]same conclusion is drawn with during same way process Niu Jingzi in 2000.Horse saussurea involucrata etc. ^[53](2002) by after the semen washing process of pig, in electric field (in 37 DEG C of environment, square topped pulse, pulsewidth 100nS, repetition rate R10Hz, amplitude 187V, sample place place field intensity is about 20V/C) showing that the transient state electricimpulse of low amplitude can form electroporation to Boar spermatozoa cell by Trypan Blue experimental result, the aperture of its perforation can import the macromole of DNA class.

Yuan Jin etc. ^[54](2007) foreign gene is injected SPF level male mouse of kunming testis fine tube, wherein one group is carried out electroporation process, and result confirms that electroporation treatment group transgenic mice positive rate is significantly higher than without electroporation treatment group.

Sperm infection protocol in 5.2 bodies

Kim J H etc. ^[17](1977) with after liposome-DNA complex transfection of spermatogonial cell, then by these spermatogonium microinjections in the convoluted seminiferous tubule of the male sterile mouse testis of artificial property, the sperm of foreign gene-carrying can after found that mouse rehabilitation, be produced.Shen Xinming etc. ^[55](2002) be directly expelled in the convoluted seminiferous tubule of mouse by foreign gene, the mating of a few Zhou Houyu female mice, obtain newborn mouse 382 altogether, PCR detects positive mice 133, and Southern blotting analyzes positive 15.Zhao Jun etc. ^[56](2003) after people Bcl-2 cDNA and mouse whey acid protein (WAP) 5 ' upstream regulatory sequence being merged, after mixing by a certain percentage with liposome, be expelled in the convoluted seminiferous tubule of 3 mouse testis, after 4 days with to oestrus female mouse and cage mating, finally obtain positive mouse 2 (1 ♂+1 ♀), Western blot confirms, genetically modified female mice mammary gland expression Bcl-2 albumen, and obtains positive F1 generation mouse.Gao Huaying etc. ^[57](2003) the recombination plasmid containing human lactoferrin gene of liposome injects rabbit testis tissue and sheep vas deferens, after one month respectively with normal doe and normal ewe mating.Success obtains transgene rabbit and goat, and detect through PCR-Southern, young rabbit and goat transgenic positive rate are 35% (11/31) and 33.3% (4/12).

Xiao Hongwei etc. ^[49](2006) directly ready injecting lipid body-hCD59 gene composite is got to testis, successfully obtain the positive pig of PCR that positive rate is 4.3% (3/73).

Foreign DNA is injected directly in buck testis by intratesticular injection method exactly, the spermatid of each stage of growth of transfection, the sperm of transfection adopts the modes such as natural crossing, hand mating or artificial insemination (AI) to obtain transgenic animal after reaching maturity in body.Intratesticular injection makes exogenous origin gene integrator to sperm and then obtains early stage positive embryos and positive offspring has been reported ^{[41,49,54,58 ~ 61]}.Li Fubing etc. ^[62]in 4 goat testis, inject pEGFP-N1, find transfection efficiency the highest generation the 40th day after injection, transfection positive rate is up to 81%, through acquisition positive rate in vitro fertilization up to 66.7% transgenic embryos.

6 ovocyte gene transfers

As a kind of ovocyte of totipotent cell, along with differentiation and the fetal development of cell, genome wherein can be expanded in all cells of organism whole genetic information ^[63].If foreign gene transfection can be entered ovocyte in prefecundation, and external source is incorporated in its genome, and through the spilting of an egg until bud into ripe individuality, each cell just can foreign gene-carrying, thus avoids the generation of chimeric animal.Chan A.W.(1998) etc. ^[12]when utilizing the ovocyte of slow-virus transfection ox, confirm that ovocyte is at the end of second time mitotic division, its nuclear membrane has breaking of short period of time, and foreign gene can enter the core of ovocyte under lentivirus mediated.This utilizes ovocyte to open a new way as transgene carrier for people, and the problem of the ovocyte nuclear membrane of long-standing problem people is solved.Carballada R etc. ^[64](2000) in vitro transfection ovocyte time, confirm that the method for liposome-mediated foreign gene is a kind of simple and effective transgenic approach.Cabot etc. ^[13](2001) by the porcine oocytes of vitro maturation, with retrovirus as carrier transfection green fluorescent protein (Green fluorescent protein, GFP) obtain positive piggy by embryo transfer, result shows that direct transfection ovocyte can obtain transgenic progeny.

Wu Desheng (2004) etc. ^[65]foreign DNA that is rear for the digestion of the ovocyte of maturation and liposome is hatched altogether, find positive signal after testing, confirm that Golden Hamster ovocyte can by the foreign DNA transfection of liposome, namely Golden Hamster ovocyte can as the carrier of foreign gene, and this result is that people utilize ovocyte production transgenic animal to provide sufficient rationale.

Laurema etc. ^[66]prove that the archeocyte core primary oocyte not having zona pellucida to protect is easily by the DNA plasmid transfection of adenovirus and liposome; and the secondary oocyte having zona pellucida to protect is not easily by the DNA plasmid transfection of adenovirus and liposome, illustrates that zona pellucida can hinder entering of foreign DNA.China Agricultural University Wang Ji expensive (2007) ^[67]establish the method that ovary injection method produces transgenic animal, and apply for patent, Yang et al. (2007) ^[68]injection of ovary green fluorescence protein gene directly to mouse, also can obtain transgenic mice, and find the transgenic mice obtained within 6 generations all there is good genetic stability.

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Summary of the invention

In view of all inconvenience and the shortcoming of above-mentioned technology, the invention provides a kind of method preparing transgenic animal of convenient operation.

The method preparing transgenic animal provided by the invention, is characterized in that, start formation stages at blastocyst, foreign gene is injected its intrauterine by direct 1ml syringe, and embryo absorbs foreign gene naturally, after natural pregnancy, normal farrowing, obtains the transgenic animal of inheritance stability.

The working concentration of wherein said foreign gene is 0.5mg/ml-0.8mg/ml, and consumption is 0.25ml, and described foreign gene comprises goal gene.

With the integration positive rate of the transgenic mice prepared by method of the present invention and rabbit all more than 70%, and the success ratio preparing the most frequently used microinjection of transgenic mice is only about 20%.

The invention provides a kind of application utilizing transgenic animal prepared by aforesaid method, it is characterized in that the object of prepared Mammals for all transgenic researches, wherein mainly comprise: the function research energy of the foundation of disease animal model, bio-reactor, gene therapy and gene.

Use method of the present invention, can be produced some and can produce the transgenic animal with business and/or pharmaceutical value product.

Use the present invention, can produce the model of some animals as humans and animals disease and other pathological states, through researchs such as diagnosis, pharmacological agent tests, be the clinical clinical testing data providing reliable diagnosis basis and reliable medicine.

Transgenic animal are produced with the present invention, do not use expensive micrurgy instrument, do not need to cultivate a large amount of ovocytes and embryo, do not need the embryo operation personnel that are skilled in technique, operation is simple for method, avoids vitro culture and the transplanting of numerous and diverse embryo.

The present invention thoroughly does away with following principle to propose: blastocyst starts formation stages, still in zona pellucida, but it is later then overflow in zona pellucida, growth to film close in sub-merit be connected before, the embryo of ungulate also will break up through further growth, trophocyte breaks up further becomes cuboidal epithelium, i.e. trophectoderm, and it can by organic and mineral solution domestic for intrauterine device and moisture absorption in segmentation cavity.

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.

Embodiment 1: the preparation of transgenic mice

Get 6 weeks ripe healthy female mouse 10 of rheological properties, after Estrus synchronization, female mouse is mated raising with the ratio of 1:1 and the normal male mouse of homology, observe mating time.After mating 48 hours, by the dosage of every gram of body weight 0.0001g, abdominal injection vetanarcol narcotic, is waiting for that the time of anesthesia prepares plasmid composite simultaneously.After the anesthesia reaction that female mouse is had a convulsion, by prostrate for female mouse Baoding.To enter female mouse back after comatose state from head about 5cm place dorsomeson zygomorphy unhairing below, expose operative site.Sterilization skin, respectively cortex and muscle layer are opened, with ophthalmic tweezers pull-out white adipose pad, namely ovary comes out, be the foreign gene pIRES2-EGFP 0.25ml of 0.5mg/ml-0.8mg/ml by working concentration with 1ml syringe, be expelled to intrauterine, inject and completely sprinkle appropriate mycillin mixture in wound, sew up a wound, again sprinkle mycillin at epidermis.

Farrow 78, after testing, 58 vivo carryings have pIRES2-EGFP gene.

Embodiment 2: the preparation of transgene rabbit

Multiparity new zealand female rabbit 6, Superovluation scheme: point 6 neck subcutaneous injection FSH, every minor tick 10 ~ 12h, injected dose is successively decreased, and total dose is 60 IU, 12h auricular vein injection hCG 100 IU after final injection FSH.With male rabbit mating after final injection hCG, join again once after 12 hours.After 48 hours of second time breeding, doe is placed on Shelf for keeing through new II anesthesia of Soviet Union's dormancy, last does routine operation to 5cm-7cm place under the intersection point of nipple and hunter's line along hunter's line otch, be the foreign gene pIRES2-EGFP 1ml of 0.5mg/ml-0.8mg/ml by working concentration with 1ml syringe, be expelled to intrauterine, inject and completely sprinkle appropriate mycillin mixture in wound, sew up a wound, again sprinkle mycillin at epidermis.

Farrow 46, after testing, 34 vivo carryings have pIRES2-EGFP gene.

Claims (6)

1. prepare a method for transgene mammal, it is characterized in that: inject in the animal uterus of post-coitum by foreign gene within the specific time period, foreign gene transfecting embryonic, normally farrows, and obtains the transgenic animal of genetic stability.

2. the method for claim 1, it is characterized in that: to the animal of normal post-coitum, start formation stages at blastocyst, directly with 1ml syringe, foreign gene is injected its intrauterine, embryo absorbs foreign gene naturally, and the final transgenic animal obtaining inheritance stability.

3. method as claimed in claim 1 or 2, is characterized in that: described animal refers to Mammals.

4. method as claimed in claim 1 or 2, it is characterized in that: the working concentration of described foreign gene is 0.5mg/ml-0.8mg/ml, consumption is that lactation animal size is determined.

5. method as claimed in claim 1 or 2, is characterized in that: described foreign gene comprises goal gene.

6. method as claimed in claim 1 or 2 prepares the application of transgene mammal, it is characterized in that: the function research prepared animal being used for disease model foundation, bio-reactor, gene therapy or gene.