CN101519664B - Method for breeding genetically modified animal by using recombinant adenovirus vector - Google Patents

Abstract

The invention relates to a method for breeding a genetically modified animal by using a recombinant adenovirus vector. The invention adopts a technical proposal that: a replication-defective adenoviral framework plasmid vector carries a foreign target gene sequence to produce a recombinant adenovirus vector in a packaging cell (293, 911 and other cells) containing a sequence of an Ela region; the recombinant adenovirus vector is combined with a donor egg or fertilized egg cell; and the donor egg or fertilized egg cell is first cultured and developed in vitro and then transferred into the body of a receptor animal for normally breeding so as to produce the genetically modified animal which is genetically stable and has functions of different target genes. The method for breeding the genetically modified animal is high in success rate. The genetically modified animal can be used in the confirmation of functional genes, the expression of pharmaceutical proteins (including organs, blood and mammary glands), and the building of models of genetically modified animal having functions of different target genes and animal genetic diseases.

Description

A kind of method of breeding genetically modified animal by using recombinant adenovirus vector

Technical field

The invention belongs to cytobiology, molecular biology and gene engineering technology field, especially a kind of is the method for preparing carriers transgenic animal with the adenovirus.

Background technology

Arrival along with the life sciences genome times afterwards comprehensively; Center on the research of gene and products thereof and use the extensive concern that causes researcher all the more; And the transgenic animal technology is wherein the most effective a kind of technological means, at present promptly handles germ cell, ovum and sperm from three aspects respectively at the strategy of transgenic animal aspect preparing in the world and obtains transgenic animal.Unstability in view of ovum and sperm processing method; So in the transgenic animal preparation; For the germ cell facture remain the most effectively, classic methods, existing just to the existing transgenic animal method for preparing, being that object handles is summarized as follows to germ cell.

Protokaryon phase microinjection foreign DNA method (Gordon JW; Scangos GA; Plotkin DJ; BarbosaJA, Ruddle FH.Genetic transformation of mouse embryos by microinjectionof purified DNA.Proc Natl Acad Sci U S is Dec A.1980; 77 (12): 7380-4.).This method is comparatively successful on mice, but extremely low for large animal (cattle, sheep, pig etc.) success rate, is merely 0.3～1%.Although there are some researcheres that the method has been done a little improvement, some progress have been obtained, like United States Patent (USP) 4736866 (1984) and 4873191 (1986).But there is inevitable defective in this method: need utilize micromanipulation system; Foreign DNA is injected accurately the male pronucleus of germ cell; It unavoidably can cause damage to body early embryo, and exogenous origin gene integrator efficient is indefinite at random, and whole efficiency is very low.It is higher that this has all caused this legal system to be equipped with the transgenic animal cost, as prepare about 500,000 dollars of transgenic cow needs.

The embryonic stem cell technology be after the protokaryon microinjection by one of transgenic technology of broad research, this method is utilized the totipotency that has of embryonic stem cell, both can develop into the ability of body one by one; After changing the reorganization embryonic stem cell that separate to obtain over to exogenous gene; Directly move into receptor, obtain transgenic animal, in view of this; The crucial part of this method is at first will set up by embryonic stem cell line, is convenient to obtain the positive embryonic stem cell of target gene in addition.But so far; Have only the embryonic stem cell of mice to succeed to build to be, and have European patent to set up external Embryonic Stem Cell Line system, method to prepare transgenic animal (WO2008017704:METHOD OF PRODUCTION OF TRANSGENIC AVIAN USING EMBRYONICSTEM CELLS).And other large animals are difficult to obtain embryonic stem cell line like pig, cattle, sheep, so the preparation of restriction large animal transgenic animal.The syncaryon implantation technique can make the deficiency that solves the embryonic stem cell source; A lot of successful research report (Mach á ty Z have been obtained in recent years; Bondioli KR; RamsoondarJJ, Fodor WL.The use of nuclear transfer to produce transgenic pigs.Cloning Stem Cells.2002; 4 (1): 21-7.), still, this method cell screening operation element in early stage amount is big, finally is easy to generate chimera, thereby has increased the cost of transgenic animal preparations.

One of most critical link is the importing of exogenous gene in the transgenic animal preparation, and glass tubing direct injection the earliest is a kind of easy method, but very big to the germ cell damage, and expensive instrument of action need and complicated operation; So the carrier that directly contains exogenous gene through structure is one of a kind of very effective means.Although cell introduction method commonly used is a lot; But because the particularity of germ cell, it has very thick cell membrane, i.e. the zona pellucida tissue; So method (like calcium phosphate method, electric shocking method, liposome method) commonly used is difficult to its operation, the target gene of achieving success imports.In recent years, the virus transfection technology is considered to the highest external source target gene leading-in technique of transfection efficiency, can realize the importing of 100% target gene.Mainly be to use the retrovirus method to prepare transgenic animal in present virus transfection method; Retrovirus is a RNA viruses; Need under the effect of reverse transcriptase, at first to change RNA into cDNA, again at dna replication dna, transcribe, a viroid that protease effect such as translation is increased down.In recent years, design construction becomes some defective viruses (defective virus) to make retrovirus become useful genophore, has successfully changed the external source target gene over to human body cell, and in cell, has expressed.Retroviral many characteristics make it become the excellent selection of transgene carrier.The most important be it can effectively be integrated into the target cell genome and express stably and lastingly with exogenous gene.Viral genome is integrated with the mode of swivel base, and its genome can not reset, and therefore entrained exogenous gene can not change yet.Although retrovirus once was widely used in the preparation of human gene therapy and transgenic animal; But this method has its limitation; At first; The exogenous gene size that this system allows to pack into is restricted, and just retroviral vector system packing exogenous gene is limited in one's ability, can not surpass 4Kb; Secondly, the target gene of this system's packing requires to contain intron sequences, thereby has limited effective regulating and expressing of target gene; The most important thing is to generate the wild-type virus granule easily after retrovirus is incorporated into host chromosome, also can activate proto-oncogene, cause host's safety issue.

Adenovirus (adenovirus) is the wire double-stranded DNA virus, and 14 genes are arranged in the genome.About 90～the 100nm of adenovirus size, the icosahedral distrand DNA virus of no outer embrane.Because adenovirus vector has high-efficient transgenic efficient and better security characteristics, has been applied to clinical nearly 20 years time of human gene therapy.Adenovirus is brought in the germ cell so can be applied to exogenous gene because it can infect non-division and somatoblast, is incorporated into host cell chromosome then.Yet; Relevant adenovirus vector prepares the transgenic animal aspect and reports that but seldom rarely seen Japanese scholar Tsukui etc. has reported that it utilizes adenovirus vector to carry out preparation research (Tsukui T, the Miyake S of transgenic mice; Azuma S; Ichise H, Saito I, Toyoda Y.Gene transfer and expression in mouse preimplantation embryosby recombinant adenovirus vector.Mol Reprod Dev.1995 Nov; 42 (3): 291-7.), yet in this research, used reporter gene LacZ fails to detect expression in the gestational age mice of transgenic progeny 13.5d.In research subsequently; This group has obtained the transgenic progeny of mice; And explored first and utilized adenovirus vector to carry out probability (the Tsukui T that transgenic animal prepare; Kanegae Y, Saito I, Toyoda Y.Transgenesis by adenovirus-mediated genetransfer into mouse zona-free eggs.Nat Biotechnol.1996Aug; 14 (8): 982-5.).Japan Patent (JP; 09-140292; A) disclose simultaneously and use CAG promoter and LacZ to be the recombinant adenovirus AxCANLacZ of reporter gene method for preparing, and method has only obtained to detect the male transgenic mice of LacZ with Southern blotting hybridization thus as the transgenic mice of carrier.In sum, at present in the world, do not find a kind of transgenic method of simple and effective.

Summary of the invention

To problem that exists in the prior art and defective, the object of the present invention is to provide a kind of method of breeding genetically modified animal by using recombinant adenovirus vector.

The technical scheme that realizes the foregoing invention purpose is a kind of method of breeding genetically modified animal by using recombinant adenovirus vector; It is characterized in that, will contain inside and outside the recombinant adenovirus granule direct infection non-human mammal germ cell of people's Antithrombin III target gene, through laboratory external fertilization and embryo culture; Early embryo is identified and screening; Acquisition has the body early embryo of genetic stability, is implanted into receptor then, obtains the transgenic animal with target gene function of genetic stability.

Described egg follicle is drawn together the germ cell of natural mating or artificial insemination acquisition, and the germ cell of external fertilization acquisition.

Described animal is meant inhuman mammal or birds.

Described target gene comprises the functional gene and the protein coding gene of non-encoding proteins, can be used for all genes of interest that in vitro transcription is expressed, and comprises polypeptide class, antibody, cytokine and enzyme etc.

Integration positive rate with the prepared transgenic mice of this method, pig, sheep, rabbit can reach more than 60%, far above traditional microinjection.

Thus, we are carrier with the recombinant adenovirus AdCMVatIII that makes up, and are example with people's Antithrombin III, have carried out the preparation of transgenic cow, sheep, pig, rabbit, and have explored the method for a kind of easier, high efficiency transgenic animal.Prepared transgenic animal can be applicable to research and application such as disease model, bio-pharmaceuticals and gene function conclusive evidence.

Description of drawings

Fig. 1 recombined human Antithrombin III adenovirus vector construct collection of illustrative plates.

Fig. 2 recombined human Antithrombin III gene adenovirus vector is identified electrophoresis pattern.

The microscopic examination figure that Fig. 3 recombinant adenovirus is produced in cell.

Fig. 4 recombinant adenoviral vector is taken people's Antithrombin III and is imported pig germ cell.

The specific embodiment

The specific embodiment of giving below in conjunction with the inventor is done further method of the present invention and is illustrated, but is to be understood that these instances only to be used to the present invention is described and is not used in restriction scope of the present invention.

Embodiment 1 people's Antithrombin III transgenic animal method for preparing

(1) synthetic, the clone of target protein (like people's Antithrombin III) cDNA and sequencing:

1. the extraction of the total RNA of human liver tissue

(1) cuts the refrigerated tissue of a fritter, promptly move in the freshly prepared lysis buffer after the weighing fast, add lysis buffer 1ml amount by every 50mg tissue.

(2) hepatic tissue is carried out the homogenate broken cell, pre-filtering is once centrifugal, collects and considers fluid.

(3) add the equal-volume chloroform, the vibration mixing, supernatant is drawn in centrifugal back.

(4) add equal-volume 70% ethanol, mixing gently, the total RNA of centrifugal acquisition.

(5) the water dissolution deposition of the no Rnase that handled with 0.01%DEPC, conventional method is carried out assay.

2. reverse transcription method is synthesized total cDNA

(1) get the total RNA of 1～2 μ g mammary gland, add water to the 9.5 μ l that DEPC handles, 70 ℃, 5～10min deposits on ice.

(2) 50 μ L reaction systems: 2.5 μ l OligodT (20pmol/L), 5 μ L, 10 * RNA PCRbuffer, 10 μ l MgCl ₂(25mmol/L), 5 μ L dNTP (each 10mmol/L), 1.25 μ L Rnase inhibitor (40U/ μ L), 2.5 μ L AMV reverse transcription (5U/ μ L) add the total RNA after 9.5 μ L handle.

(3) reaction condition: 30 ℃, 10min, 42 ℃ of 15min, 99 ℃ of 5min, 4 ℃ of 10min, product be stored in-70 ℃ subsequent use.

3. utilize polymerase chain reaction (PCR) amplification hATIII cDNA

(1) is example with people's Antithrombin III, designs following special primer and be used for pcr amplification that making the primer final concentration is 20 μ M.

(2) forward primer: 5 '-TCA GGTACCATGTATTCCAATGTGATAG-3 ' (line place is the KpnI restriction enzyme site)

Downstream primer: 5 '-TCTCTCGAGCTACTTAACACAAGGGTTG-3 ' (line place is the XhoI restriction enzyme site)

Add continuously in (3) the 100 μ L reaction systems: 10 * PCR buffer, 10 μ L, dNTP (each 2.5mmol/L) 16 μ L, forward primer 1 μ l, downstream primer 1 μ l, synthetic cDNA product 2 μ L in 2 add dH ₂O to 100 μ L.

(4) PCR reaction condition: 94 ℃ of preparatory degeneration 5min, 97 ℃ of degeneration 30s, 60 ℃ of annealing 30s, 72 ℃ are extended 2min, 30 circulations, 72 ℃ are extended 10min again.Product be stored in-70 ℃ subsequent use.

4.PCR product purification

With hATIII PCR product purification, use commercial test kit, like the PCR of BIOTECH company product fast purifying Kit.

(1) the standard agar electrophoresis is separated above-mentioned PCR product, and agarose concentration is 1%, 100V voltage stabilizing state, electrophoresis 40min.

(2) under uviol lamp, accurately cut the segment about DNA size 1.4Kb on the gel, and weigh.

(3) by the test kit requirement, certain proportion adds the colloidal sol buffer, in 37～55 ℃ of colloidal sols, during do not stop to rock.

(4) sample is on the absorption pillar behind the interpolation colloidal sol, and adsorption of DNA is with Washing Buffer rinsing 2 times.

(5) add dH ₂O is in the absorption pillar, and centrifugal eluted dna is in-20 ℃ of preservations.

5. the cloning of target gene hATIII

With target gene hATIII PCR purified product with KpnI and XhoI enzyme action after, use simultaneously the pUC19 cloning vehicle carried out coupled reaction after with SalI and the KpnI enzyme action that can produce identical sticky end.

Coupled reaction: 1 μ L PCR purified product, 0.5 μ LpUC19 plasmid, 0.5 μ L T ₄DNA Ligase enzyme, 1 μ L, 10 * T4 DNA Ligase Buffer, 7 μ L dH ₂O.16 ℃ of night incubation.

Antibacterial transforms: get the above-mentioned coupled reaction product of 5～10 μ l, use conventional CaCl ₂Chemical method, thermostimulation method transformed competence colibacillus DH5 α.

Transformed bacteria is plated on ammonia benzyl resistance (100 μ g/mL Amp ^r) the solid bacteria culture plate, 37 ℃ of cultivations.

Next day, picking list bacterium colony is inoculated in 3～5mL liquid LB culture fluid, 37 ℃ of insulation 10～15h.

The little upgrading grain of conventional alkaline lysis, the restricted enzyme cutting analysis recombiant plasmid.

6.hATIII gene sequencing

The reorganization positive plasmid that restricted enzyme cutting analysis is correct is delivered to gene sequencing company and is carried out gene sequencing behind the purification.This gene order in Shanghai outstanding auspicious biological engineering (GENERAY) company limited accomplished order-checking; Analysis shows to sequencing result to use general in the world NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) gene order software; The target gene sequence is (like people's Antithrombin III among sequence of being cloned and the international GENBANK; ACCESSION:NM_000488) unanimity, its complete sequence is shown in sequence table SEQ 1.TXT.

Automatically translated by biosoftware, the recombiant protein that the present invention produced in conjunction with instantiation, is that example is 464 peptides with human thrombin III, and its sequence is following, and preceding 32 peptides of sequence are the signal peptide section, shown in sequence table SEQ 2.TXT.

(2) target gene albumen (is example like people's Antithrombin III gene) construction of recombinant adenovirus containing and viral vector production

Target gene cDNA sequence (people's antithrombase cDNA sequence) is connected into carrier, after sequence verification, is built into target recombinant albumen (like people's antithrombase) adenovirus vector jointly with adenovirus skeleton plasmid carrier.With restricted enzyme Kpn1 and Xho1 double digestion hATIIIcDNA sequence, same enzyme action vector plasmid (as, pShuttle-cmv), connect, transform by (one) 5 step, obtain recon (like pShuttle-cmv-hATIII), see Fig. 1.

Plasmid recon (pShuttle-cmv-hATIII) is made up acquisition restructuring lactoferrin adenovirus vector AdhLTF with the skeleton plasmid that contains the adenoviral gene group, see Fig. 1.And through restriction endonuclease analysis, the genomic correctness of checking recombinant adenovirus is seen Fig. 2, and (A) pcr amplification is identified AdCMVatIII, (B) digestion with restriction enzyme recombinant adenovirus genome, 1: recombinant adenovirus genome AdCMVatIII among Fig. 2; 2: the adenoviral gene group that does not contain target gene; 3: not enzyme-added recombinant adenovirus genome.

In HEK293,911 cell lines, produce viral vector and a large amount of amplification with recombinant human protein's gene (like people's Antithrombin III gene) adenovirus vector AdCMVatIII.Through the GFP reporter gene can the spike target protein expression efficiency, like observed result under (A) simple microscope among Fig. 3, (B) observed result under the fluorescence microscope, the target protein expression efficiency is very high.

Cesium chloride gradient centrifugation obtains to be used for zooperal highly purified recombinant adenovirus granule, carries target gene, like people's Antithrombin III, and AdCMVatIII virus titer 10 ¹²More than the PFU/mL.

Select the eukaryotic cell expression system for use; Like CHO (Chinese hamster ovary cell), sheep mammar gland epithelial cell, rabbit, cattle, pig galactophore epithelial cell etc., the vivoexpression efficient of utilizing detection recombinant adenoviral vectors such as RT-PCR, Western blotting to carry target gene.

(3) recombinant human protein's gene (like people's Antithrombin III gene) adenovirus vector infection animal fertilized egg cell

With recombinant human protein's gene (like people's Antithrombin III gene) adenovirus vector infection animal fertilized egg cell, In vitro culture, fluorescence microscope, screening obtains the positive embryo that target gene is transcribed and expressed.

(1) 2～4h after the mice natural mating obtains protokaryon phase germ cell; With adenovirus AdCMVatIII, titre 2～8 * 10 ⁸PFU/mL is hatched altogether.

(2) porcine oocytes after maturation in vitro is cultivated, carries out external fertilization with pig bright smart (kind pig farm, Changli, Hebei) from slaughterhouse, Changli, Hebei, receives precision processing 1～2h; Adenovirus AdCMVatIII, titre 0.8～1 * 10 ⁹PFU/mL is hatched altogether, with the germ cell of AdCMVatIII recombinant virus particle infected pigs, 24～48 hours, detects transcribing and expressing of target gene.The positive pig embryo ectogenesis that obtains through screening is implanted into the pseudo-fetus sow to blastula stage, normal farrowing, and Southern blot method detects target gene and whether is incorporated into host genome, and Western blot detects whether normal expression of target gene.Like Fig. 4 abc, wherein A: immature porcine oocytes; B: the porcine oocytes that is used for external fertilization; C: transgenic positive pig embryo, pig embryo under the fluorescence microscope behind the adenovirus infection 48h.

(3) bovine oocyte is from local slaughterhouse, and external oocyte is cultivated ripe, through external fertilization; Adenovirus AdCMVatIII, titre 0.8～1 * 10 ⁹PFU/mL is hatched altogether.

(4) sheep is raised back HORMONE TREATMENT superovulation from local farmers', and ovum pick-up after the natural mating obtains germ cell; Adenovirus AdCMVatIII, titre 0.8～1 * 10 ⁹PFU/mL is hatched altogether.

(5) rabbit is raised back HORMONE TREATMENT superovulation from the local farmers' in ox head precipice, Changli, Hebei, and this friendship back modus operandi is adopted ovum, obtains germ cell; Adenovirus AdCMVatIII, titre 0.8～1 * 10 ⁹PFU/mL is hatched altogether.

(4) detection of transgenic animal and evaluation

Conventional Sorthern blotting method detects the integration of target gene group in animal (Mus, pig, cattle, sheep, rabbit) chromosome, and Western Blotting method detects the expression of target gene in the transgenic animal tissue.Used one anti-is the anti-target protein of rabbit (Mus) (like people's Antithrombin III) monoclonal antibody, and two anti-ly are the anti-rabbit of HRP HRPO labelling (Mus) Ig, and target protein (like people's Antithrombin III) standard substance are the Sigma product.

Be to be understood that in addition; After having read above-mentioned teachings of the present invention; Those skilled in the art can do various changes or modification to the present invention, and various forms transgenic animal, transgenic animal model and the gene protein medicine that produces with the method or technology belongs under the application within claims institute limited range equally.

< 110>Xibei Univ. of Agricultural & Forest Science & Technology

< 120>a kind of method of breeding genetically modified animal by using recombinant adenovirus vector

<160>2

<210>1

<211>464

<212>DNA

< 213>people's Antithrombin III

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ATGTATTCCAATGTGATAGGAACTGTAACCTCTGGAAAAGGGAAGGTTTATCTTTTGTCC60

TTGCTGCTCATTGGCTTCTGGGACTGCGTGACCTGTCACGGGAGCCCTGTGGACATCTGC120

ACAGCCAAGCCGCGGGACATTCCCATGAATCCCATGTGCATTTACCGCTCCCCGGAGAAG180

AAGGCAACTGAGGATGAGGGCTCAGAACAGAAGATCCCGGAGGCCACCAACCGGCGTGTC240

TGGGAACTGTCCAAGGCCAATTCCCGCTTTGCTACCACTTTCTATCAGCACCTGGCAGAT300

TCCAAGAATGACAATGATAACATTTTCCTGTCACCCCTGAGTATCTCCACGGCTTTTGCT360

ATGACCAAGCTGGGTGCCTGTAATGACACCCTCCAGCAACTGATGGAGGTATTTAAGTTT420

GACACCATATCTGAGAAAACATCTGATCAGATCCACTTCTTCTTTGCCAAACTGAACTGC480

CGACTCTATCGAAAAGCCAACAAATCCTCCAAGTTAGTATCAGCCAATCGCCTTTTTGGA540

GACAAATCCCTTACCTTCAATGAGACCTACCAGGACATCAGTGAGTTGGTATATGGAGCC600

AAGCTCCAGCCCCTGGACTTCAAGGAAAATGCAGAGCAATCCAGAGCGGCCATCAACAAA660

TGGGTGTCCAATAAGACCGAAGGCCGAATCACCGATGTCATTCCCTCGGAAGCCATCAAT720

GAGCTCACTGTTCTGGTGCTGGTTAACACCATTTACTTCAAGGGCCTGTGGAAGTCAAAG780

TTCAGCCCTGAGAACACAAGGAAGGAACTGTTCTACAAGGCTGATGGAGAGTCGTGTTCA840

GCATCTATGATGTACCAGGAAGGCAAGTTCCGTTATCGGCGCGTGGCTGAAGGCACCCAG900

GTGCTTGAGTTGCCCTTCAAAGGTGATGACATCACCATGGTCCTCATCTTGCCCAAGCCT960

GAGAAGAGCCTGGCCAAGGTGGAGAAGGAACTCACCCCAGAGGTGCTGCAGGAGTGGCTG1020

GATGAATTGGAGGAGATGATGCTGGTGGTCCACATGCCCCGCTTCCGCATTGAGGACGGC1080

TTCAGTTTGAAGGAGCAGCTGCAAGACATGGGCCTTGTCGATCTGTTCAGCCCTGAAAAG1140

TCCAAACTCCCAGGTATTGTTGCAGAAGGCCGAGATGACCTCTATGTCTCAGATGCATTC1220

ATAAGGCATTTCTTGAGGTAAATGAAGAAGGCAGTGAAGCAGCTGCAAGTACCGCTGTTG1280

TGATTGCTGGCCGTTCGCTAAACCCCAACAGGGTGACTTTCAAGGCCAACAGGCCTTTCC1320

TGGTTTTTATAAGAGAAGTTCCTCTGAACACTATTATCTTCATGGGCAGAGTAGCCAACC1380

CTTGTGTTAAGTAG1396

<210>1

<211>464

<212>DNA

< 213>people's Antithrombin III

<400>1

MYSNVIGTVTSGKGKVYLLSLLLIGFWDCVTCHGSPVDICTAKPRDIPMNPMCIYRSPEK60

KATEDEGSEQKIPEATNRRVWELSKANSRFATTFYQHLADSKNDNDNIFLSPLSISTAFA120

MTKLGACNDTLQQLMEVFKFDTISEKTSDQIHFFFAKLNCRLYRKANKSSKLVSANRLFG180

DKSLTFNETYQDISELVYGAKLQPLDFKENAEQSRAAINkWVSNKTEGRITDVIPSEAIN240

ELTVLVLVNTIYFKGLWKSKFSPENTRKELFYKADGESCSASMMYQEGKFRYRRVAEGTQ300

VLELPFKGDDITMVLILPKPEKSLAKVEKELTPEVLQEWLDELEEMMLVVHMPRFRIEDG360

FSLKEQLQDMGLVDLFSPEKSKLPGIVAEGRDDLYVSDAFHKAFLEVNEEGSEAAASTAV420

IAGRSLNPNRVTFKANRPFLVFIREVPLNTIIFMGRVANPCVK464

Claims (1)

1. the method for a breeding genetically modified animal by using recombinant adenovirus vector; It is characterized in that, will be by the AdCMVatIII adenovirus particles direct infection non-human mammal germ cell of structure shown in Figure 1, through embryo culture; Early embryo is identified and screening; Acquisition has the body early embryo of genetic stability, is implanted into receptor then, obtains the transgenic animal with target gene function of genetic stability.

Images