CN1730659A - Method for preparing mammal galactophore biological reactor using gene targeting technology - Google Patents

Method for preparing mammal galactophore biological reactor using gene targeting technology Download PDF

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Publication number
CN1730659A
CN1730659A CN 200510055256 CN200510055256A CN1730659A CN 1730659 A CN1730659 A CN 1730659A CN 200510055256 CN200510055256 CN 200510055256 CN 200510055256 A CN200510055256 A CN 200510055256A CN 1730659 A CN1730659 A CN 1730659A
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gene
gene targeting
protein
animal
targeting
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袁三平
张义静
鲜建
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QINGDAO SENMIAO BIOTECHNOLOGY INSTITUTE
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QINGDAO SENMIAO BIOTECHNOLOGY INSTITUTE
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Abstract

The invention discloses a method for preparing mammals lactiferous gland bioreactor through gene targeting technique which comprises the following steps: (1) constructing medical protein genes, Lox sequence, Ploy(A) signal, screening genes and lox sequences into the expression frame of the milk protein genes of mammals to construct gene targeting vectors, (2) transforming the host cells and screening, (3) obtaining gene targeting cell strains through PCR and Southern analysis, (4) transplanting nucleus of the gene targeting cell strains, obtaining gene targeting cloned animal, (5) multiplying the gene targeting animal, (6) transferring the Cre protein into the fertilized ovum or somatic cells of the gene targeting sheep for re-cloning, obtaining gene targeting animal with no screening genes, (7) adding the second medical protein gene into gene targeting animal, thus obtaining a gene targeting bioreactor capable of synthesizing two medical protein genes.

Description

Utilize gene targeting to prepare the method for mammal galactophore biological reactor
Technical field
The present invention relates to gene engineering, specifically, belong to a kind of method of utilizing gene targeting to prepare mammal galactophore biological reactor.
Background technology
The medical protein gene is the extrinsic protein gene that is used for the treatment of respective egg white matter disappearance or lacks, thousands of protein gene is arranged in the organism, they are all carrying out corresponding special function, some protein gene is indispensable in organism or keeps certain protein level, otherwise will cause disease.Industrial protein is industrial important raw and processed materials, as silk and spider silk fibroin or the like.The intensity of spider silk fibroin and elasticity is good than steel all, and its is much more in light weight, is referred to as biological steel, is space flight, military project and civilian important materials.
Producing protein with mammal galactophore biological reactor is to produce one of medical and industrial proteinic important method.Its significance is that galactophore biological reactor and fermentation using bacteria and cell cultures produce medical protein matter and compare, and have the following advantages: the kinds of protein in (1) milk is few, and it is convenient to extract; (2) cost is low, only needs a conventional animal to raise, and does not need the device of fermentation using bacteria and cell cultures jar etc.; (3) mammary gland synthetic protein has glycosylation, the biological activity height.
From 1997 the somatic cell clone sheep " many jasmines " cultivate successfully since (Wilmut et al, 1997), mammal galactophore biological reactor becomes the focus of scientific research and social concerns again.Compare with the transgenic animal that procaryotic injection obtains, the somatic clone of large animal transgenosis has following advantage: obtain the efficient height of transgenic animal, fund input is few; The more important thing is, can be at first the cell of vitro culture be carried out genetic modification, as genetically modified integration and detection, the inactivation of genomic gene and the fixed point of foreign gene are inserted (gene targeting, also claim gene site-directed integration, and then obtain the cloned animal of corresponding transgenosis and gene targeting homologous recombination).
Gene targeting (gene targeting) is an important Protocols in Molecular Biology that grows up the eighties in 20th century, be to utilize gene transfer method, after exogenous DNA array imported target cell, by the reorganization between homologous DNA sequence on the karyomit(e) in exogenous DNA array and the target cell, the foreign DNA site-directed integration is gone into a certain definite site on the target cell genome, or a certain target site that pre-determines carried out rite-directed mutagenesis, thereby change the method for cytogenetics characteristic.As emerging technology, gene targeting has unrivaled advantage: the cell that gene targeting was suitable for promptly can be a prokaryotic cell prokaryocyte, also can be eukaryotic cell; Gene targeting can be introduced foreign gene on the specific fragment of chromosomal DNA; Under situation reasonable in design, gene targeting can carry out meticulous transformation to the host cell chromosome gene; Behind the gene targeting by the gene that hit or the new gene of introducing with chromosomal DNA duplicate and stable duplicating.This technology is being transformed biological variety biological, that cultivation is new at present, studies gene mechanism and function, expresses and regulation and control, and the aspects such as gene therapy of research regulatory mechanism cell life cycle and inherited disease are applied and are constantly perfect.
First has reported transgenic and cloned animal (sheep) Schnieke etc. (1997); (Baguisi etal, 1999) clone goat is succeedd after 2 years; Keefer equals acquisition transgene clone goat in 2002; Inst. of Genetics and Development Biology, CAS etc. have also reported clone goat and the transgene clone goat (Zou et al, 2001 and 2002) in the Yangzhou birth respectively in 2001 and 2002.The gene targeting of large animal works in and began that report was arranged in 2000, and McCreath etc. arrive somatic a1 collagen protein protogene (a1-procollagen) site with medical protein plasmagene site-directed integration, and have obtained clone sheep; Denning etc. (2001) report is by a1 galactosyltransferase (a1-galactosyltransferase) gene and the Prion protein gene inactivation of gene targeting with sheep.(2002) such as Lai etc. and Dai have almost reported the research success of gene targeting pig (galactosyltransferase gene inactivation) at one time.But domestic and international up to now seldom useful gene targeting prepares the report of lactation galactophore biological reactor.Part is used at random, and the product of the mammalian biological reactor of transgenosis preparation has entered clinical experimental stage.For example: the plain III of the people's that U.S. GTC biotherapy company produces antithrombin has entered clinical experimental stage; The spider silk fibroin of Canada Nexia biotech company has begun to develop in large quantities and produce.So, adopt gene targeting to prepare the lactation galactophore biological reactor and will further improve the content of medical protein matter in milk.
The Laoshan milk goat is one of high yield milk goat of China, milk yield is big, and the phase of giving milk, the elder can reach 10 months more than 8 months, one tire sheep average year is given milk 310 kilograms, two tire sheep average years are given milk 590 kilograms, and the triplet average year is given milk more than 700 kilograms, and Gao Zheke reaches 1300 kilograms, the milk yield that is equivalent to 1/3 cow head, and the research cycle of goat is short, and the feeding and management expense is low, is one of important research object of galactophore biological reactor.The plain III gene of the people's that applicant is used antithrombin, its gene product are the key proteins (Lu etal, 2000) of human health care and treatment some diseases.The plain III of antithrombin is a kind of plasma glycoprotein, the hemostasis of it and wound and keep the mobile closely related of body inner blood.Clinically, the plain III of antithrombin is mainly used in the treatment myocardial infarction, thrombophlebitis, plain deficiency disease of zymoplasm and thrombosis (cerebral thrombosis) etc.And be used for surgical procedures, enter blood circulation (Levy et al, 2002, Konkle et al, 2003) to prevent and to reduce the coagulation of blood piece.
The patent application publication number of Shanghai Genon Bioengineering Co., Ltd be disclose in the patent application of CN1502694 a kind of by the method for Cre-loxp fixed point target practice somatic cell clone domestic animal as galactophore biological reactor, this method at first changes gene targeting carrier in the somatocyte over to, clone, obtain cloned animal or clone body cell, and then the medical protein gene is transferred in clone's the somatocyte, carry out the somatic clone of cloned animal again one time, obtain genetically modified gene targeting animal at last.Promptly need to carry out twice somatic cell gene and transform, screening and somatic cell clone, the cycle that obtains gene targeting mammary gland of domestic animal bio-reactor just almost doubles, and has strengthened workload and the cost of producing medical protein.
Summary of the invention:
The objective of the invention is to overcome the deficiencies in the prior art part, with medical or industrial protein gene, method by gene targeting, with the functional gene site-directed integration to the site of mammiferous mammary gland protein gene, the expression of endogenous mammary gland protein gene is obstructed, and express the functional gene that changes over to, and form composition with the protein that changes in the milk, increase the expression amount of functional gene.Produce medical or industrial proteinic cost thereby reduce, be the mankind's Health and Living service.
Technical solution of the present invention is: utilize gene targeting to prepare the method for mammal galactophore biological reactor, comprise the following steps:
With medical protein gene, Lox sequence, Ploy (A) signal, screening-gene and lox sequence construct in the expression framework of mammiferous milk protein gene, make up gene targeting carrier;
2. transformed acceptor cell and screening;
3. analyze by PCR and Southern, obtain the cell strain of gene targeting;
4. the nuclear transplantation of the cell strain of gene targeting obtains the gene targeting cloned animal;
5. the expansion of gene targeting animal is numerous;
6. Cre protein is changed in the zygote of gene targeting sheep or the i.e. clone again of somatocyte, obtain not have the gene targeting animal of screening-gene;
7. utilize the characteristics of Cre-lox and IRES sequence, second medical protein gene is added in the gene targeting animal, obtain to synthesize the gene targeting bio-reactor of two medical protein genes.
Beneficial effect of the present invention is as follows: the present invention compares with mammary gland bioreactor of transgenic animals, and its obvious benefit is: the medical protein expression of gene amount of gene targeting is higher than genetically modified expression amount (approximately will be higher than 2-10 doubly).And sharp Poly (A) signal of mammary gland protein gene, reduced the production cost of medical protein on the one hand, be that enterprise increases profit, on the other hand, reduce the selling price on the market, be patient's service.
CN1502694 compares with Chinese patent application, and the gene targeting carrier of this patent has a medical protein plasmagene
The complete ceneme of (people source Antithrombin III gene) (Poly (A) signal that the growth hormone gene of an ox is arranged in the back of people source Antithrombin III gene), gene targeting carrier is expressed medical protein matter in the mammary gland of the cloned animal of the first-generation, shortened the time of the cloned animal galactophore biological reactor that obtains medical protein gene gene targeting, simultaneously, this gene targeting carrying agent, in the somatic cell clone animal of random integration, also be a kind of genetically modified at random bio-reactor; The method of CN1502694 patent application need be carried out twice somatic cell gene and be transformed, screening and somatic cell clone, and the cycle that obtains gene targeting mammary gland of domestic animal bio-reactor just almost doubles, and has strengthened workload and the cost of producing medical protein.Two Lox sequences are arranged in the gene targeting carrier of this patent, they are same directions, under the proteinic effect of Cre, screening-gene (Neo) is excised, just can utilize Poly (A) signal of beta-casein gene, increase medical protein expression of gene amount, reduce the cost of whole bio-reactor.On remaining unique lox site, utilize the characteristics of Cre-lox reorganization and IRES sequence, also can add second medical protein gene, two or more medical protein genes are expressed in same gene targeting animal simultaneously efficiently.In the method for CN1502694 patent application, the medical protein plasmagene does not utilize Poly (A) signal of beta-casein gene.
Description of drawings
Fig. 1: the structure of milk goat beta-casein gene and gene targeting route synoptic diagram.
Fig. 2: the cell strain of gene targeting and Southern of clone sheep and pcr analysis.
Among the figure: the 1st, the gene targeting clone sheep; 2, the 4th, the fetus of gene targeting becomes fine minicell strain, and the 3rd, control group sheep DNA, W is a blank, and MW is a 1KB dna molecular amount mark (Invitrogen), and DNA cuts with the Pst1 enzyme, and the DNA between Pst1 and the Kpn1 is a probe.
Fig. 3: the raising up seed of first gene targeting clone sheep.
Among the figure: gene targeting clone ram and offspring's PCR and Southern result:
1, a, b, c, d, e, f, g, h, I, j, k, l, m, n, o, p, q, r, s are the offsprings of gene targeting clone ram, and t is the DNA sample of gene targeting ram itself, and the 5.0kb segment is the genomic fragment of goat, and 6.0kb is the gene targeting segment, and MW is the dna molecular amount.
2, PCR is with all gene targeting clone rams and offspring's DNA sample thereof, but has only the DNA of PCR male sheep to be used for Southern hybridization.
The analysis of Fig. 4, the second batch of gene targeting sheep and Southern and PCR.
Among the figure: acquisition and PCR and the Southern analytical results of new gene targeting clone ram:
AT3.1, AT3.2 and AT3.3 are the clone sheep that obtains from the gene targeting cell strain, and AT3.1 and AT3.2 are the gene targeting sheep, and AT3.3 does not contain the gene targeting segment, prove the clone sheep that comes from normal cell.C is normal sheep DNA contrast, and the 5.0kb segment is the genomic fragment of goat, and 6.0kb is the gene targeting segment, and MW is the dna molecular amount.
Embodiment:
Describe the present invention in detail below in conjunction with drawings and Examples.
The present invention includes following steps:
(1) selects mammiferous dairy-protein basis for use because genetically modified control region and expression framework, on the expression framework of milk protein gene, set up a unique restriction enzyme site, then the medical protein gene is connected, on the expression framework of milk protein gene, the screening-gene of gene targeting carrier, Poly (A) signal and medical protein gene connect together, and are separated by by two unidirectional Lox sequences.
(2) constructed carrier is determined the dna sequence dna of functional gene and milk protein gene through dna sequence analysis, and forms consistent with the expression of milk protein gene itself and the amino acid of functional gene.
(3) with electroporation or liposome-induced method genophore is changed in the somatocyte.
(4) the pair cell strain is screened in substratum, the positive cell strain after obtaining to screen; Part cell freezing is preserved, and a part of cell is used to prepare DNA.
(5) DNA of positive cell strain through PCR and Southern hybridization analysis, determines the cell strain of homologous recombination (gene targeting).
(6) cell of Chong Zu cell strain moves in all cracks of ovum of sophisticated enucleation oocyte, and donor cell and enucleation oocyte are merged.
(7) with clone embryo transplantation in the foster mother sheep, obtain the gene targeting clone sheep.
(8) emulsion of collection gene targeting animal, purification medical protein matter.
(9) (clone again) in the zygote or somatocyte with Cre albumen transgene target practice animal, the excision screening-gene.
(10) with in second medical protein plasmagene transgene target practice animal gene group.
In operating process, can utilize the Cre-lox technology that screening-gene is excised from gene targeting carrier; And second functional gene fixed point is added on the lox site of the somatocyte of gene targeting sheep or zygote, prepare the gene targeting galactophore biological reactor (as Fig. 1) of new functional gene fast.
Be example with the Laoshan milk goat below, adopt antithrombin factor gene (Antithrombin III, ATIII, AT3) make up that (β-casein) is the gene targeting carrier of framework, and finally to obtain with the antithrombin factor gene be the Laoshan milk goat galactophore biological reactor of functional gene with the goat casein gene.Referring to Fig. 1, its specified operational procedure is as follows:
(1) acquisition of functional gene
The Antithrombin III gene is to obtain mRNA from people's related tissue, is transcribed into cDNA, sets up the connection restriction enzyme site at two ends then by PCR method.The PCR product is connected on the pUC19, carries out dna sequence analysis again; The dna sequence dna of clone's ATIII gene is identical with disclosed dna sequence dna.Concrete grammar is: the preparation of Antithrombin III gene is DNA sequence (the Gene Bank according to disclosed ATII, D29832), design PCR primer (and adding 1 unique Xho1 enzyme restriction enzyme site before primer) carries out the PCR reaction, the PCR product cloning is to the pUC19 carrier, carry out sequence analysis, the dna fragmentation of again that the DNA sequence analysis is correct ATIII, add a lox sequence, the PolyA sequence, neomycin gene (Neomycin) and Lox sequence are connected on the expression framework of milk goat beta-casein gene dna vector at last.
(2) structure of gene targeting carrier
Segment with the beta-casein gene of sheep is a probe, from the genomic library of Laoshan milk goat, isolate one section complete beta-casein gene, and with PCR method remove part second exon of beta-casein gene and the 7th exon and between dna sequence dna, set up a Not1 site, with Antithrombin III and Poly (A) signal and the neomycin gene gene that contain two same direction Lox sequences, be connected on the expression framework of beta-casein gene then.
(3) transformed acceptor cell
Isolate fetal fibroblast the Laoshan milk goat fetus of from 30 to 45 days gestational ages, isolated cells is cultivated at DMEM-F12 (1: 1) and is added in the substratum of 10% foetal calf serum (FCS) and antibiotic.Gene targeting carrier is changed in the goat fetal fibroblast in the substratum that contains G418 (600ug/ml) the about 15-20 of screening and culturing days then with electroporation (Bio-rad) or liposome mediated-method; Cell strain expands numerous again, and a part of cell freezing is preserved, and a part is used to prepare DNA.
Present embodiment is divided into the fetus from 6 Laoshan milk goats, sets up 6 initiating cell systems.Therefrom screened 4 clones and be used for cell transfecting, gene targeting carrier has been changed in the fetal fibroblast; Screening is about 15-20 days in the DMEM-F12 that contains 600ug/ml G418 (1: 1) substratum.In the screening process of gene targeting cell, cell is grown, and cell strain is all selected preferably, so the surviving rate of cell strain is low, and show after the DNA analysis, have only about 1% cell strain that the homologous recombination of gene has taken place, finally have only 0.3% cell strain to can be used for clonogenic assay, see Table one.
The growth of table one cell and transgenosis and gene targeting efficient
The genophore type The test multiplicity The cell strain number The cell strain number of DNA analysis Positive cell number The cell strain number that can be used for cloning
AT III gene targeting 3 1293 541 6 4(0.3%)
(4) nuclear transplantation of positive cell strain
Goat is handled through estrus synchronization and super row, obtains the ovocyte and the foster mother sheep of cylinder mature.Positive cell is after external hungry 2-5 days, move in all cracks of ovum of enucleation oocyte, method with electricity irritation merges donor cell and enucleation oocyte, after the vitro culture 4 hours, activate 5 minutes with ionomycin (Ionomycin), in the substratum that contains 6-xylidine purine (6-DMAP), cultivated 5 hours again.At last, clone embryos moves in the normal substratum and cultivates; Second day, move in the foster mother sheep uterine tube.
(5) preparation of DNA, Southern and pcr analysis
The tissue of cultured cells and a small amount of clone sheep is changed in the dna cleavage liquid (containing Proteinase K), in 55 ℃ of water-baths, spend the night, add the straight alcohol of twice then, mixing, centrifugal; Wash DNA once with 70% alcohol, seasoning adds an amount of TRIS-EDTA solution then, treats that DNA all after the dissolving, puts into-20 ℃ refrigerator.Spend the night with Pst1 enzymic digestion dna solution, electrophoresis on 0.8% gel changes DNA in the tunica fibrosa over to then, hybridizes with the P32 label probe respectively.Obtain the Southern picture of DNA.With the PCR primer cell strain and the clone sheep of gene targeting carried out pcr analysis.
The clonogenic assay of cell carries out in two batches.After donor cell is recovered from liquid nitrogen, vitro culture for some time, in containing the substratum of 0.5%FCS hungry 2-5 days then.The ovocyte of cylinder mature is for supplying ovum.Obtain 313 ovocytes altogether, fusion rate is: among 78%, 174 piece of clone embryo transplantation to 22 foster mother, and the pregnancy of 1 foster mother sheep, 1 clone of output lamb.Southern and pcr analysis show: the clone lamb contains the Antithrombin III gene, first clone goat that contains the gene targeting of medical protein gene that this is in the world to be obtained.
The nuclear transplantation statistic data sees Table two, and figure two is seen in Southern of gene targeting sheep and the analysis of PCR.Among the figure two, the cell strain of gene targeting and the Southern of clone sheep thereof and pcr analysis figure.The 1st, the gene targeting clone sheep; 2.4 the fetus that is gene targeting becomes fine minicell strain, and the 3rd, control group sheep DNA, W is a blank, and MW is that 1KB dna molecular amount mark (Invitrogen) .DNA cuts with the Pst1 enzyme, and the DNA between Pst1 and the Kpn1 is a probe; The PCR condition is: 94 2 minutes; Then 94 ℃ 25 seconds, 65 ℃ 15 seconds, 72 8 minutes, totally 31 the circulation; Following 10 minutes at 68 ℃ at last.
Table two nuclear transplantation cartogram
The cell clone date: The 10-11 month in 2002 The 10-11 month in 2003
The ovocyte of cylinder mature: 313 161
Ovocyte and donor cell complex body 221 141
The fusion cloning embryo: 174 97
The transplanting embryo number: 86 97
Foster mother sheep number: 22 8
60 days conceived sheep numbers: 1 5
Conceived sheep number to childbirth: 1 3
Litter size: 1 3
Survive the clone sheep number: 1 3
To so far, first gene targeting clone sheep (male) that contains the Antithrombin III gene grows normally, and with other Laoshan milk goat mating, obtained offspring's (seeing its offspring's PCR and Southern analysis chart) than the gene targeting sheep of large group.Growing of three gene targeting clone sheep that are born again in March, 2004 is normal.Analyze through PCR and Southern, it is clone sheep of gene targeting that two clone sheep are wherein arranged, and another is the clone sheep of cloning from normal cell.These two gene targetings clone rams with the female sheep mating in relevant milk mountain, g and D is all good.
Sequence table
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<120〉utilize gene targeting to prepare the method for mammal galactophore biological reactor
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1 GGTACCTGAT GTCATCTTAA ATTGCTGGCT TTTTGATTTT CCATTGGACA 50
AGCTTCTTTC TTTAGTATAT TGTTAAGGAT TTCCTTGATC AAGATTTTAC 100
CTACTTTTCT GGTCCAATTG GTGAGAGACA GTCATAAGGA AATGCTGTGT 150
TTATTGCACA ATATGTAAAG CATCTTCCTG AGAAAATAAA AGGGAAATGT 200
5 TGAATGGGAA GGATATGCTT TCTTTTGTAT TCCTTTTCTG AGAAATCAGA 250
CTTTTTCACC TGTGGCCTTG GCCACCAAAA GCTAACAAAT AAAGGCATAT 300
GAAGTAGCCA AGGCCTTTTC TAGTTATATC TATGACACTG AGTTCATTTC 350
ATCATTTATT TTCCTGACTT CCTCCTGGGT CCATATGAGC AGTCTTAGAA 400
TGAATATTAG CTGAATAATC CAAATACATA GTAGATGTTG ATTTGGGTTT 450
10 TCTAAGCAAT CCAAGACTTG TATGACAGTA AGATGTATTA CCATCCAACA 500
CACATCTCAG CATGATATAA ATGCAAGGTA TATTGTGAAG AAAAATTTTT 550
AATTATGTCA AAGTGCTTAC TTTAGAAGGT CATCTATCTG TCCCAAAGCT 600
GTGAATATAT ATATTGAAGG TAATGAATAG ATGAAGCTAA CCTTGTAAAA 650
ATGAGTAGTG TGAAATACAA CTACAATTAT GAACATCTGT CACTAAAGAG 700
15 GCAAAGAAAC TTGAAGATTG CTTTTGCAAA TGGGCTCCTA TTAATAAAAA 750
GTACTTTTGA GGTCTGGCTC AGACTCTATT GTAGTACTTA GGGTAAGACC 800
CTCCTCCTGT ATGGGCTTTC ATTTTCTTTC TTGCTTCCCT CATTTGCCCT 850
TCCATGAATA CTAGCTGATA AACATTGACT ATAAAAGATA TGAGGCCAAA 900
CTTGAGCTGT CCCATTTTAA TAAATCTGTA TAAATAATAT TTGTTCTACA 950
20 AAAGTATTAT CTAAATAAAT GTTACTTTCT GTCTTAAAAT CCCTCAACAA 1000
ATCCCCACTA TCTAGAGAAT AAGATTGACA TTCCCTGGAA TCACAGCATG 1050
CTTTGTCTGC CATTATCTGA CCCCTTTCTC TTTCTCTCTT CTCACCTCCA 1100
TCTACTCCTT TTTCCTTGCA ATTCATGACC CAGATTCACT GTTTGATTTG 1150
GCTTGCATGT GTGTGTGCTG AGTTGCGTCT GACTGTTATC AACCCCATGA 1200
25 ATGATAGTCC ACCAGGCTCT ACTGTCCATG AAATTTTCCA GTCAAGAATA 1250
CTGGAGTGGA TTGCATTTCC TACTCCATTT GATTAATTTA GTGACTTTTA 1300
AATTTCTTTT TCCATATTCG GGAGCCTATT CTTCCTTTTT AGTCTATACT 1350
CTCTTCACTC TTCAGGTCTA AGGTATCATC GTGTGCTTGT TAGCTTGTTA 1400
CTTTCTCCAT TATAGCTTAA GCACTAACAA CTGTTCAGGT TGGCATGAAA 1450
30 TTGTGTTCTT TGTGTGGCCT GTATATTTCT GTTGTGTATT AGAATTTACC 1500
CCAAGATCTC AAAGACCCAC TGAATACTAA AGAGACCTCA TTGTGGTTAC 1550
AATAATTTGG GGACTGGGCC AAAACTTCCG TGCATCCCAG CCAAGATCTG 1600
TAGCTACTGG ACAATTTCAT TTCCTTTATC AGATTGTGAG TTATTCCTGT 1650
TGAAATGCTC CCCAGAATTT CTGGGGACAG AAAAATAGGA AGAATTCATT 1700
35 TCCTAATCAT GCAGATTTCT AGGAATTCAA ATCCACTATT GGTTTTATTT 1750
CAAACCACAA AATTAGCATG CCATTAAATA CTATATATAA ACAGCCACTA 1800
AATCAGATCA TTATCCATTC AGCTTCTCCT TCACTTCTTC TCCTCTACTT 1850
TGGAAAAAAG GIAAGAATCT CAGATATAAT TTCAGTGTAT CTGCTACTCA 1900
TCTTTATTTT GGACTAGGTT AAAATGTAGA AAGAACATAA TTGCTTAAAA 1950
40 TAGATCTTAA AAATAAGGGT GTTTAAGATA AGGTTTACAC TATTTTCAGC 2000
AGATATGTTA AAAAATAGAA GTGACTATAA AGACTTGATA AAAATTATAG 2050
TGACTGCAAA TGTTTTAGGA ATATAATAAG ATATAATAAC AGTGGTTGCT 2100
ATTTTCTTTA GCACAAGACT AGTCAACAGG CTGTATTAAA AGATCTTTTC 2150
TTGAATTAAA TATTTTCAAT TTGATTAAAC CTACCTCAGC CATAAAGGCA 2200
45 AGCACATTTC ATTTATACTA TGGGGATTTG AATAATTATT ACTGAAGAAG 2250
CTCTACCAAC AAAAAGTTTA TAGAGCTATC ATATTTAGTC AAGAGATAAA 2300
GAGGGTTGTT AGGATATATA TGCTATTTGA AAGGTATTTA TAAAAGAAGA 2350
GTATATTTAT CAAAATTTCT CAGAACATCC AAATTTCAAG TTTATCATTT 2400
ATCTTACAAT ATTTCAAAAA TATTAAAATA GATACATGAA ATACAGAAGT 2450
50 AAATTAAAGA GAAAGTATTT TACTTGGTAA AAAAATTCTA GGTTGGACAG 2500
AGAGTGCCAG GAAACAAAAA CAATGAAAAA TGTGACCTGA CTGGAATTAT 2550
AGCTCAAAGT ATAGTAGTAA GTAATGAAAT GGCTTAAAAA TTGGTATATA 2600
AAATGCTAGT TATAAAATAA ACAAAATGCA ATAATATCCT CCCTACATGT 2650
AATGAATTCT AGGTATTATG CTCTTTTTTG AAGTCTTGAC AATAAAAATT 2700
55 TTTTTAGAAG TTTATAGGCA TCTTGAATAA AGTGAAACAA ATTAAGAATT 2750
AGTATCCATG AGAAAAATAT AGAACAATTT TCCTAATTTA GTTTGAAAAT 2800
CTGGGATTGA AGATGTGTGT CAAGAGATGT TGGTGGCAAG AACATTTTTT 2850
TTTCAAGAAC TTATAAAAAT GCAACAAAAC AAACCATTTA ATACATTTTG 2900
GTCAAAATCA ATAATGTATT TTATTTTATG CTCCAAGGAG CATAAAATTG 2950
60 GGGACTGGGC AAGAGAAACT GACACCCTGG TAAATTACCA AGAGATAAGT 3000
ACACAGTTCT ATGTAGAGAA AATAAGCATA GTGTATGATC TCTAAAATTA 3050
TGTGAGACAA AGGAGAGATG ACATTAGGCA TGTGGGGATG AAGACTGAGT 3100
AGAGAAGAAA CAATCTAATC AGTCCAAGAA AACATCTCGA TCAGTGGAAC 3150
AAATAGAAGA AATGCTAAAA TGAAACAGAA GTCTTACTGG AAATAAAAGA 3200
65 TATGCATAAG ACAAAAATTC ATGAAAATCA CTTAGTTTAG CAGAGAAAAG 3250
ATAAAAATAA AGTATGACCT TCTTCATATA CATTGTTTGA TCATATGCAC 3300
CTCAATAAAA CTGAGTCTCC AACAGAAATG AAACATTAAT ATTTTGTTCA 3350
CTGCTCTAAT CCCAGAATCT AAGCGATATC TGGCAATAAA AATAATAAAT 3400
ATATATTTTT TAATAAATGA ATCAACCACT TAATTTTTCT GTAAATATCT 3450
70 GTAACTTCTC TTCTGTCTTT CCAAAAACAC TCATAAGTAC TGTGAATGAG 3500
ATGAAAAAGA GTGAAGTAGG ATATAGGCTG TTAGCAGAAA ACATCTGAAT 3550
GGCTGGCAGT GAAACATTAA CTTGAAATGT AAGATTAATG AGTAATAGTA 3600
AATTTTAACC TTGGCCATAT GATAAAATGT TCATTAATAT TTTTCTAGAA 3650
TACAGGGCTT TTTGTTTTTG CCATGAGGTT TGCAGGATCT TGGTTCCCTG 3700
75 ACCAGGGATC AAACCTGCAC TCCCCTGGAA GCATGGAGTC TTGGACATCT 3750
GTATTATACA CTATCTTTGG TTCCTTTTAA AGGGAAGTAA TTTTACTTAA 3800
ATAAGAAAAT AGATTGACAA GTAATACGCT GTTTCCTCAT CTTCCCATTC 3850
ACAGGAATCG AGAGCC 3866
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<223〉protein secreting sequence
<400>3
1 ATGAAGGTCC TCATCCTTGC CTGTCTGGTG GCTCTGGCCA TTGCA 45
<210>4
<211>6
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(6)
<223〉Xho1 site
<400>4
1 CTCGAG 6
<210>5
<211>15
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(15)
<223〉Enterokinase protease cutting site
<400>5
1 GACGACGACG ACAAG 15
<210>6
<211>1398
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1398)
<223〉people source Antithrombin III gene
<400>6
1 ATGATTTATT CCAATGTGAT AGGAACTGTA ACCTCTGGAA AAAGGAAGGT 50
TTATCTTTTG TCCTTGCTGC TCATTGGCTT CTGGGACTGC GTGACCTGTC 100
ACGGGAGCCC TGTGGACATC TGCACAGCCA AGCCGCGGGA CATTCCCATG 150
AATCCCATGT GCATTTACCG CTCCCCGGAG AAGAAGGCAA CTGAGGATGA 200
5 GGGCTCAGAA CAGAAGATCC CGGAGGCCAC CAACCGGCGT GTCTGGGAAC 250
TGTCCAAGGC CAATTCCCGC TTTGCTACCA CTTTCTATCA GCACCTGGCA 300
GATTCCAAGA ATGACAATGA TAACATTTTC CTGTCACCCC TGAGTATCTC 350
CACGGCTTTT GCTATGACCA AGCTTGGTGC CTGTAATGAC ACCCTCCAGC 400
AACTGATGGA GGTATTTAAG TTTGACACCA TATCTGAGAA AACATCTGAT 450
10 CAGATCCACT TCTTCTTTGC CAAACTGAAC TGCCGACTCT ATCGAAAAGC 500
CAACAAATCC TCCAAGTTAG TATCAGCCAA TCGCCTTTTT GGAGACAAAT 550
CCCTTACCTT CAATGAGACC TACCAGGACA TCAGTGAGTT GGTATATGGA 600
GCCAAGCTCC AGCCCCTGGA CTTCAAGGAA AATGCAGAGC AATCCAGAGC 650
GGCCATCAAC AAATGGGTGT CCAATAAGAC CGAAGGCCGA ATCACCGATG 700
15 TCATTCCCTC GGAAGCCATC AATGAGCTCA CTGTTCTGGT GCTGGTTAAC 750
ACCATTTACT TCAAGGGCCT GTGGAAGTCA AAGTTCAGCC CTGAGAACAC 800
AAGGAAGGAA CTGTTCTACA AGGCTGATGG AGAGTCGTGT TCAGCATCTA 850
TGATGTACCA GGAAGGCAAG TTCCGTTATC GGCGCGTGGC TGAAGGCACC 900
CAGGTGCTTG AGTTGCCCTT CAAAGGTGAT GACATCACCA TGGTCCTCAT 950
20 CTTGCCCAAG CCTGAGAAGA GCCTGGCCAA GGTAGAGAAG GAACTCACCC 1000
CAGAGGTGCT GCAAGAGTGG CTGGATGAAT TGGAGGAGAT GATGCTGGTG 1050
GTCCACATGC CCCGCTTCCG CATTGAGGAC GGCTTCAGTT TGAAGGAGCA 1100
GCTGCAAGAC ATGGGCCTTG TCGATCTGTT CAGCCCTGAA AAGTCCAAAC 1150
TCCCAGGTAT TGTTGCAGAA GGCCGAGATG ACCTCTATGT CTCAGATGCA 1200
25 TTCCATAAGG CATTTCTTGA GGTAAATGAA GAAGGCAGTG AAGCAGCTGC 1250
AAGTACCGCT GTTGTGATTG CTGGCCGTTC GCTAAACCCC AACAGGGTGA 1300
CTTTCAAGGC CAACAGGCCT TTCCTGGTTT TTATAAGAGA AGTTCCTCTG 1350
AACACTATTA TCTTCATGGG CAGAGTAGCC AACCCTTGTG TTAAGTAA 1398
<210>7
<211>34
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(34)
<223〉loxp sequence
<400>7
1 ATAACTTCGT ATAGCATACA TTATACGAAG TTAT 34
<210>8
<211>6
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(6)
<223〉Taq1 site
<400>8
1 CTCGAC 6
<210>9
<211>420
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(420)
<223〉Poly of bovine growth hormone gene (A) signal sequence
<400>9
1 ATTAATACGA CTCACTATAG GGAATTCCAG GGGGGTGGTG GATATCCTGG 50
TACCGCGGCC GCTCGAGCAT GCATCTAGAG GGCCCTATTC TATAGTGTCA 100
CCTAAATGCT AGAGCTCGCT GATCAGCCTC GACTGTGCCT TCTAGTTGCC 150
AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC CCTGGAAGGT 200
5 GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG CATAAGCTTG 250
GCACTGGCCG TCGTTTTACA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC 300
CCAACTTAAT CGCCTTGCAG CACATCCCCC TTTCGCCAGC TGGCGTAATA 350
GCGAAGAGGC CCGCACCGAT CGCCCTTCCC AACAGTTGCG CAGCCTGAAT 400
GGCGAATGGA AATTGTAAGC 420
<210>10
<211>6
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(6)
<223〉Xho1 site
<400>10
1 CTCGAG 6
<210>11
<211>1104
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1104)
<223〉Neo gene order
<400>11
1 ATCCGAACAA ACGACCCAAC ACCCGTGCGT TTTATTCTGT CTTTTTATTG 50
CCGATCCCCT CAGAAGAACT CGTCAAGAAG GCGATAGAAG GCGATGCGCT 100
GCGAATCGGG AGCGGCGATA CCGTAAAGCA CGAGGAAGCG GTCAGCCCAT 150
TCGCCGCCAA GCTCTTCAGC AATATCACGG GTAGCCAACG CTATGTCCTG 200
5 ATAGCGGTCC GCCACACCCA GCCGGCCACA GTCGATGAAT CCAGAAAAGC 250
GGCCATTTTC CACCATGATA TTCGGCAAGC AGGCATCGCC ATGGGTCACG 300
ACGAGATCCT CGCCGTCGGG CATGCGCGCC TTGAGCCTGG CGAACAGTTC 350
GGCTGGCGCG AGCCCCTGAT GCTCTTCGTC CAGATCATCC TGATCGACAA 400
GACCGGCTTC CATCCGAGTA CGTGCTCGCT CGATGCGATG TTTCGCTTGG 450
10 TGGTCGAATG GGCAGGTAGC CGGATCAAGC GTATGCAGCC GCCGCATTGC 500
ATCAGCCATG ATGGATACTT TCTCGGCAGG AGCAAGGTGA GATGACAGGA 550
GATCCTGCCC CGGCACTTCG CCCAATAGCA GCCAGTCCCT TCCCGCTTCA 600
GTGACAACGT CGAGCACAGC TGCGCAAGGA ACGCCCGTCG TGGCCAGCCA 650
CGATAGCCGC GCTGCCTCGT CCTGCAGTTC ATTCAGGGCA CCGGACAGGT 700
15 CGGTCTTGAC AAAAAGAACC GGGCGCCCCT GCGCTGACAG CCGGAACACG 750
GCGGCATCAG AGCAGCCGAT TGTCTGTTGT GCCCAGTCAT AGCCGAATAG 800
CCTCTCCACC CAAGCGGCCG GAGAACCTGC GTGCAATCCA TCTTGTTCAA 850
TGGCCGATCC CATATTGGCT GCAGGGTCGC TCGGTGTTCG AGGCCACACG 900
CGTCACCTTA ATATGCGAAG TGGACCTGGG ACCGCGCCGC CCCGACTGCA 950
20 TCTGCGTGTT CGAATTCGCC AATGAAACCA CACTGCTCGA CATTGGGTGG 1000
AAACATTCCA GGCCTGGGTG GAGAGGCTTT TTGCTTCCTC TTGCAAAACC 1050
ACACTGCTCG AGGAATTCAT AACTTCGTAT AATGTATGCT ATACGAAGTT 1100
ATGC 1104
<210>12
<211>34
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(3866)
<223〉Lox sequence
<400>12
1 ATAACTTCGT ATAGCATACA TTATACGAAG TTAT 34
<210>13
<211>3866
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(9)
<223〉Not1 site
<400>13
1 GCGGCCGCC 9
<210>14
<211>2233
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2233)
<223〉part the 7th exon to the 9 exon genes sequences of milk goat beta-casein.,
3 ' homology arm of gene targeting carrier
<400>14
1 ATCCCTTCAC TGGGCCCATC CCTAACAGCC TCCCACAAAA CATCCTGCCT 50
CTTACTCAAA CCCCTGTGGT GGTGCCGCCT TTCCTTCAGC CTGAAATAAT 100
GGGAGTCCCC AAAGTGAAGG AGACTATGGT TCCTAAGCAC AAAGAAATGC 150
CCTTCCCTAA ATATCCAGTT GAGCCCTTTA CTGAAAGCCA GAGCCTGACT 200
5 CTCACTGATG TTGAAAAGCT GCACCTTCCT CTGCCTCTGG TCCAGTCTTG 250
GATGCACCAG CCTCCCCAGC CTCTTTCTCC AACCGTCATG TTTCCTCCTC 300
AGTCCGTGCT GTCCCTTTCT CAGCCCAAAG TTCTGCCTGT TCCCCAGAAA 350
GTAGTGCCCC AGAGAGATAT GCCCATCCAG GCCTTTCTGC TGTACCAGGA 400
GCCTGTACTT GGTCCTGTCC GGGGACCCTT CCCTATTCTT GTAAGTCTAA 450
10 ATTTACTAAC TGTGCTGTTT AACTTCTGAT GTTTGTATGA TATTTGAGTA 500
ATTAAGAGCC CTACAAAAAA TCAATAATGA ATGGTTCCAA AATAAGCATA 550
GCTGAGATTA ATGATTCTCA GCATTGGTTA TAAATAGAAT AAGCTGGAAA 600
ACCTTCACCT CCCCTCCACC ACCAGATCTC AATGTCTAGG CTTACCCATG 650
GAGATTCTGA TTAACTGTTC TTTCTATGTA GAAGAAACTT ATTGGGAAGA 700
15 AATAATATAA TGGACTATGA TTTAATTGGT CTGTTGAGAA TTTAGATGAA 750
GGGGATTAAG TTACAATAAA GCCAGAATTG AACTTGATAA TCTCACTTGG 800
CTAAGAATAA CAAACCTAAG AAGGTTTGCT ATTTTCTACA ATTTTGAAGT 850
TTTCCTTATG CACAATTATT TCACCACATG ACTCATTTCA CCTCTTGTTT 900
TTGATATATG AGCATATGAG GGCAAAATAC TGAAGATGCT TATTTCAATA 950
20 CTCAGGGAAA ATTTTCTTGC CAAAAGGCAA GAATTGTATA ATTCATTCAC 1000
TTATTTTATT TTTTTAATTT TTAAGGTCTA AGAGGATTTC AAAGTGAATG 1050
CCCCCTCCTC ACTTTTGGTA AGCTTTAGGA GATTGGAGGC AGACTGATCA 1100
TTTTTATAGT TAATATCTTT TACATTTCAT CTTCCTGGAT AAGCCCCAAT 1150
AGTAGCAATT TCTATCAGTA TACCAGCGTA AAGATTAGTT TTAAATATAT 1200
25 TTTCAGTGAT TGACTGTTAT TTACTGACCT GAAATTATGT ATCTGTTATA 1250
TTTCAAATAA TGCAAAACTG TATATATATG GTGTTGACAG ATTTGATTGG 1300
TTTTCTTTCA ATTGCCTATA TCCTTATTAT TGATTGTAAT CATTTATAGA 1350
AAAAACAAAA TAATTTCTTA TACTTTTATG TAAACCTGTT AGAGCTTATT 1400
TTAAAGATCA ACTGCATTCA CATTTCTAAT CTAGTCATTA TGAGCTTCAA 1450
30 TTGTTTTATC TCACTTAAAA TTTATATATT GTCTTTTAAT TCATGAGTCA 1500
AAATACAATC TCACAGTCCA GATATGGGAC TTAAAAGGGG AATAGCATAT 1550
AGTTTTGATA TTCTTAAAGA AATACATCTT TTTGTGATCA TGATTCAGCA 1600
GACATTTTAA TAAAACAATT CCAAGTGAGC CGACACTTGG TCCTAGAGGA 1650
ATTTTTATAA TCTTAAGGTA AGGCACAGCA TGGTGTTTTT GTAATAAGAT 1700
35 TTCTTTTATG AAAAAGTCAC ACCAAAATTG GAAATGGGGT GAGATGAAGA 1750
GTTATAACAT ATAACTAAAT GGACATTTGT TCTCTATTCC ACAGAATTGA 1800
CTGCGACTGG AAATATGGCA ACTTTTCAAT CCTTGCATCA TGCTACTAAG 1850
ATAATTTTTA AATGAGTATA CATGGAACAA AAAATGAAAC TTTATTCCTT 1900
TATTTATTTT ATGCTTTTTC ATCTTAATTT GAATTTGAGT CATAAACCAT 1950
40 ATACTTTCAA AATGTTAATT CAACATTAGC ATAAAAGTTC AATTTTAACT 2000
TGGAAATATC ATGAACATAT CAAATTATGT ATAAAAATAA TTTCTGGAAT 2050
TGTGATTATT ATTTCTTTAA GAATCTATTT CCTAACCAGT CATTTCAATA 2100
AATTAACCCT TAGGCATATT TAAGTTTTCT TGTCTTTATT ATATTTTTAA 2150
AAATGAAATT GGTCTCTTTA TTGTTAACTT AAATTTATCT TTGATGTTAA 2200
AAATAGCTGT GGAAAATTAA AATTGGATAG AAT 2233
<210>15
<211>6
<212>DNA
<213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(6)
<223〉Sma1 site
<400>15
1 CCCGGG 6

Claims (7)

1, a kind of method of utilizing gene targeting to prepare the non-human mammal galactophore biological reactor is characterized in that
This method comprises the following steps:
(1) with medical protein gene, Lox sequence, Ploy (A) signal, screening-gene and lox sequence construct in the expression framework of mammiferous milk protein gene, make up gene targeting carrier;
(2) transformed acceptor cell and screening;
(3) analyze by PCR and Southern, obtain the cell strain of gene targeting;
(4) nuclear transplantation of the cell strain of gene targeting obtains the gene targeting cloned animal;
(5) expansion of gene targeting animal is numerous;
(6) Cre protein is changed in the zygote of gene targeting sheep or the i.e. clone again of somatocyte, obtain not have the gene targeting animal of screening-gene;
(7) utilize the characteristics of Cre-lox and IRES sequence, second medical protein gene is added in the gene targeting animal, obtain to synthesize the gene targeting bio-reactor of two medical protein genes.
2, method according to claim 1 is characterized in that described milk protein gene comprises: lactoglobulin, alpha-casein gene, beta-casein gene, serum protein gene, bovine lactoferrin gene.
3, method according to claim 1 is characterized in that described medical protein gene is connected on the expression framework of milk protein gene, and site-directed integration is on endogenous milk protein gene, then specific high-efficiency expression in milk.
4, method according to claim 1 is characterized in that described Mammals comprises: ox, goat and sheep, pig, rabbit, horse.
5, method according to claim 1, it is characterized in that Poly (A) termination signal is added in the back of medical protein plasmagene, the cell strain of homologous recombination obtains the transgenic and cloned animal of gene targeting through after cloning, and directly efficiently expresses medical protein in its milk.
6, method according to claim 1 is characterized in that utilizing the Cre-lox technology that screening-gene is excised from gene targeting carrier, utilizes Poly (A) termination signal of beta-casein gene.
7, method according to claim 1 is characterized in that utilizing adding second two medical protein gene on unique lox sequence, and two medical protein genes are expressed in same animal simultaneously.
CN 200510055256 2005-03-17 2005-03-17 Method for preparing mammal galactophore biological reactor using gene targeting technology Pending CN1730659A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 200510055256 CN1730659A (en) 2005-03-17 2005-03-17 Method for preparing mammal galactophore biological reactor using gene targeting technology

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Application Number Priority Date Filing Date Title
CN 200510055256 CN1730659A (en) 2005-03-17 2005-03-17 Method for preparing mammal galactophore biological reactor using gene targeting technology

Publications (1)

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CN1730659A true CN1730659A (en) 2006-02-08

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102296088A (en) * 2010-09-07 2011-12-28 青岛森淼实业有限公司 Method for expressing human antithrombin-III gene to in mammary gland of capra hircus L.
CN103184232A (en) * 2011-12-31 2013-07-03 北京中农创新生物工程研究院有限公司 Animal cell friendly locus screening carrier and screening method

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102296088A (en) * 2010-09-07 2011-12-28 青岛森淼实业有限公司 Method for expressing human antithrombin-III gene to in mammary gland of capra hircus L.
CN103184232A (en) * 2011-12-31 2013-07-03 北京中农创新生物工程研究院有限公司 Animal cell friendly locus screening carrier and screening method

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