WO2012068769A1 - Procédé d'une intégration spécifique au site de gènes exogènes - Google Patents

Procédé d'une intégration spécifique au site de gènes exogènes Download PDF

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WO2012068769A1
WO2012068769A1 PCT/CN2011/000183 CN2011000183W WO2012068769A1 WO 2012068769 A1 WO2012068769 A1 WO 2012068769A1 CN 2011000183 W CN2011000183 W CN 2011000183W WO 2012068769 A1 WO2012068769 A1 WO 2012068769A1
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gene
casein
site
cells
human
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PCT/CN2011/000183
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Chinese (zh)
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成功
汤波
李宁
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北京济福霖生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/101—Bovine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to a method for site-directed integration of a foreign gene. Background technique
  • Lysozyme is an important antibacterial factor in human milk. It has a killing effect on most Gram-positive bacteria and some Gram-negative bacteria. It plays an important role in enhancing the immunity of infants and young children. At the same time, lysozyme is in health care. Light industry, food industry and other aspects are also widely used.
  • mammary gland bioreactor to produce recombinant protein has broad market application prospects, but traditional transgenic technology still has certain deficiencies in the study of mammary gland bioreactor.
  • traditional transgenic research since the foreign gene is a random insertion in the genome, affected by the positional effect and the size of the vector itself, the inability to contain the complete regulatory elements often makes the expression level of the foreign gene low or even Expression or ectopic expression (Clark, 1994; Dobie 1996; Kim, 2007).
  • this integration may also present a certain risk to the host itself (Seggewis, 2006; McCormack, 2004). These disadvantages greatly reduce the reliability of transgenic animal breeding.
  • Gene targeting is a transgenic technology for gene targeting.
  • a homologous recombination between homologous genes and genomic sequences is used to achieve a modified technique for site-specific modification of chromosomes. It is strong, can stabilize genetics and foreign genes are not affected by positional effects, and has no effect on neighboring genes. It will become an ideal method for transforming gene genes in the future.
  • K ⁇ McCreath used a gene targeting method to integrate the human antitrypsin gene (AAT) into the sheep collagen fragment (COL1A1). The human antitrypsin expression in goat milk was 65 ( ⁇ g/ml (McCreath).
  • transgenic use by gene targeting make the transgenic research controllable, significantly improve the success rate of transgenic animals in transgenic animal breeding, improve the safety of transgenic animals, reduce the risk of transgenic breeding, and facilitate Large-scale breeding.
  • the object of the present invention is to provide a method for efficiently and stably integrating foreign genes, which can greatly increase the expression level of foreign genes, and improve breeding success rate and safety reliability.
  • the method for site-directed integration of a foreign gene is to use a gene targeting method to integrate a human lysozyme gene or other gene of interest into a target locus of a mammalian-specific protein expressed in a mammal such as a cow, a sheep or a rabbit.
  • the lysozyme gene or other gene of interest captures intact mammary-specific expression protein regulatory sequences and regulatory sequences at the locus to achieve efficient expression of human lysozyme or other protein of interest in the mammalian mammary gland.
  • the human lysozyme gene is site-integrated into the bovine asl-casein locus by a gene targeting method, and a targeting vector constructed according to the above method is obtained, which has the structure shown in FIG.
  • Human lysozyme or other gene of interest in the mammary gland is very active in the transcription of the casein locus, overcoming the positional effect, avoiding silencing of the target gene, and achieving higher expression of human lysozyme or other recombinant protein in the mammary gland , improve the success rate of transgenic breeding, and at the same time save the cost of producing recombinant protein in the later stage;
  • Target gene integration at the chromosomal site avoids the effects of random insertion and multi-copy insertion on the expression of adjacent important genes (eg insertional mutations, multiple copies of insertions causing chromosomal instability, inactivation of adjacent genes, activation of proto-oncogenes, Inactivated tumor suppressor genes, etc.), improve the safety and controllability of genetically modified organisms, and eliminate some people's concerns about the safety of genetically modified foods;
  • the human lysozyme genome sequence can be used instead of the cDNA sequence to increase the expression level of human lysozyme.
  • Figure 1 is a result of PCR amplification of a homologous left arm in Example 1 of the present invention, wherein M: lkb marker; 1-3: homologous left arm PCR amplification product;
  • Figure 2 is a result of PCR amplification of homologous right arm in Example 1 of the present invention, wherein 1-5: Homologous right arm PCR amplification product; M: lkb marker;
  • Figure 3 is a result of PCR amplification of human lysozyme gene in Example 1 of the present invention, wherein 1-4: human lysozyme gene PCR amplification detection; M: lkb marker;
  • Example 4 is a result of enzyme digestion of LYZ-k-in targeting vector in Example 1 of the present invention, wherein 1: Sall (5597 bp + 16278 bp); 2: XhoI (6243 bp + 15632 bp); 3: Notl/S all (5597 bp + 5526 bp) +10752bp); 4: NotI/XhoI (2593bp+6243bp+13039b p); 5: HindIII (4474bp+ 17401 bp); 6: NotI(linear, 21875bp); 7: Plas mid as control; M: lkb marker;
  • Figure 5 is a schematic diagram showing the fusion of asl-casein and human lysozyme gene in Example 1 of the present invention; wherein F1, R1 and F2, R2 indicate RT-PCR detection of primer position;
  • Figure 6 is a result of RT-PCR F1R1 primer detection in Example 1 of the present invention, wherein M: lkb marker; 1-4: positive cell detection result; 5-6: untransfected C127 control; 7-8: RNA template; : H 2 0 as a template; 10: cDNA detection;
  • Figure 7 is a result of RT-PCR F2R2 primer detection in Example 1 of the present invention, wherein M: lkb marker; 1-4: F2R2 primer detection result;
  • FIG. 8A and FIG. 8B are the results of sequencing the RT-PCR primer F2R2 PCR product in the first embodiment of the present invention.
  • the DNAMAN1 sequence is the sequencing result of the PCR product
  • the DNAMAN2 sequence is the NCBI published sequence asl-casein untranslated mRNA and human lysozyme.
  • Gene fusion mRNA; the box-annotated sequences are restriction endonuclease Xhol site, lysozyme mRNA translation initiation (ATG) and translation termination sequence (TAA);
  • Figure 9 is a diagram showing the expression of lysozyme in breast cells by Western-blot in Example 1 of the present invention, wherein 1: untransfected targeting vector C127 cells were induced; 2: transfected targeting vector cells were not induced; 3: transfected targeting vector cells Induction
  • Figure 10 is a fetus on the 42nd day of pregnancy in the first embodiment of the present invention.
  • Figure 11 is a schematic diagram showing homologous recombination in Example 1 of the present invention, wherein A: casein locus; B: asl-casein gene structure map; C: gene targeting vector; D: human lysozyme integration site after homologous recombination; 3'HRF and 3'HRR primers are shown above;
  • Figure 12 is a PCR analysis of target cells in Example 1 of the present invention, wherein 1-2: cells in which homologous recombination occurs; 3: 094 genome as a negative control; 4: gene targeting vector as a control; 5: H 2 0; M: Lkb marker;
  • Figure 13 is a result of sequencing of a PCR product of a target cell in Example 1 of the present invention, wherein the underlined portion is a homologous right-arm downstream primer, and the box portion is a homologous right-arm flanking sequence (a homologous right arm in the asl-casein gene) Downstream sequence);
  • Figure 15 is a diagram showing the gene carrier of the site-integrated human lactoferrin gene in Example 2 of the present invention. detailed description
  • Example 1 Asl-casein locus site-specific integration of other target gene targeting vectors (taking human lysozyme gene as an example)
  • the upstream primer (see SEQ ID N0.1) 5 -ATTTGCGGCCGCTAAT GTTCCTGTCATACAACTGTGAAT -3' was designed according to the bovine asl-casein sequence published by GeneBank (Genesis No.: X5985 6), and the Notl restriction site was introduced; downstream primer (see SEQ ID) NO.2) 5 -CCCTCGAGGTCAAGATCTATGTAAGAA AATAAAATAG-3', introduced Xhol enzyme cleavage site, used to amplify the homologous left arm (Left arm, 9354-11939, 2586 bp).
  • the upstream primer (see SEQ ID NO.
  • the upstream primer (see SEQ ID N0.5) 5 -CCGCTCGAGAACATGAAGG CTCTCATTGTTC-3' was designed according to the human lysozyme sequence published by GeneBank (Genebank No.: X14008), and the Xhol enzyme cleavage site was introduced; the downstream primer (see SEQ ID NO. 6) 5 -CCGCTCGAGTAGAAGTGTAATATGAGGCCAG-3 ' , introduced Xhol restriction site, and amplified human lysozyme gene sequence using human genomic DNA as template, including lysozyme self-tailing signal (544-6780, 6236bp) (Fig. 3), and Link to the TOPO-T vector for sequencing verification.
  • the sequencing results showed that the amplified human lysozyme sequence exon and the cleavage site were not mutated, and the LYZ-T vector was constructed.
  • the Plox vector was used as the backbone vector (7460 bp), and the Plox vector was digested with Sail and the Right arm-T was recovered and ligated to complete the construction of the vector R-PLOX.
  • the constructed R-PLOX vector was digested with NotI and Xhol. Since the Left arm and PMD19-T vectors were close in size, the Left arm fragment was recovered by digestion with NotI, XhoI and Pvul, and then ligated into the R-PLOX vector. Construction of LPR vectors.
  • LYZ-T and L-P-R vectors were digested with Xhol to link the recovered LYZ sequence to the Xhol site of L-P-R vector, and the target vector was constructed and named LYZ-K-in.
  • the completed targeting vector was digested (Fig. 4) and sequenced (see Figure 14 for the construction process).
  • the left-arm amplification region of the gene targeting vector was as1-casein promoter region, exon I and part of exon II region, so the targeting vector was transfected into mouse C127 mammary gland.
  • the cells can detect whether the homologous left arm can guide the correct expression of human lysozyme and verify the correctness of the vector.
  • the LYZ-K-in targeting vector was linearized with Notl and then transfected into mouse breast cancer cell C127 with reference to Lipo-2000 transfection reagent. After 48 hours of transfection, the cells were passaged at 1:6 and then added with 80 ( ⁇ g/ml G418 for screening. The liquid was changed every three days, and cloned spots were formed around 7 days. The screening was continued for three days, and the cells were collected for later detection.
  • the cells obtained after screening were induced with DMEM containing prolactin, insulin and hydrocortisone for 48 hours, and the supernatant was collected and concentrated by ultrafiltration using an ultrafiltration centrifuge tube (4 ml, 3 KD) 50 times. The cells were used for RA extraction and protein. collect.
  • RNA collected by the induced cells was subjected to reverse transcription, and the reverse transcribed DNA was used as a template, and the primer was identified by LYZ: F1 (see SEQ ID NO. 7) 5 -TGC AAAGAGGGTTGTCCGTGAT-3 ' , Rl (see SEQ ID NO.8) 5 -CAAT ACTTGTAAGCTCATCTGCCTC-3' and LYZ fusion identification primer: F2 (see SE Q ID NO.9) 5'- CTTGCTGCTTCTTCCCAGTC-3 ' , R2 (see SEQ ID NO.
  • the lysozyme standard solution was set at concentrations of 100, 150, 200, 250, 300, 350, and 400 U/mL. Take a 90 mM solution of Micrococcus lysate (OD 46 ( 0.9) in a 96-well plate, quickly add the enzyme solution ⁇ , and mix the 5S with a TECAN microplate reader to measure the light absorption at 460 nm. Timing, recording every lmin, each group of data is repeated three times or more. The average value of the difference of OD 46() of 0 to 1 minute is plotted on the ordinate, and the standard enzyme concentration is used as the abscissa to make the standard curve.
  • Bovine fetal fibroblasts were established by trypsinization. The specific methods are as follows: a. Slaughter 42 days of cows, remove the placenta into DMEM medium, return to the laboratory 12h ( Figure 10). b. Remove fetal head, limbs, viscera and cartilage tissue in cell culture JUL containing 5xDPBS, place the remaining tissue in a new 10 cm cell culture J, and cut as much as possible with an ophthalmic scissors. c.
  • the cells obtained by centrifugation are inoculated into a T25 cell culture flask at a concentration of l xlO 6 /bottle, and cultured in a 37.5 ° C, 5% C0 2 incubator, and the solution is changed every two days until the cells are full. Cryopreserved and named 094 fetal fetal fibroblasts.
  • the cells were transfected and transfected with amaxa nucleofector.
  • the specific procedure is as follows: Two bottles of T25 cells (about 8 ⁇ 10 6 ), linearized targeting carrier 12 ⁇ ⁇ and 400 ⁇ l Nucleofector reagents were mixed and dispensed into 4 electric shock cups for electric shock, and then cultured in four bottles of T-25. Cultivate in a dish. After 48 h, the cultured cells were digested and plated into 60 10 cm dishes containing 50 ( ⁇ g/ml G418 and 2 M GANC medium for cell selection, and after screening for about 10 days, a total of 15 to 48 well plates were picked. After the cells were 90% confluent, they were expanded to 24-well plate culture, and 9 clones grew better. Then, the cells after half-long storage were frozen, and half were used for genome extraction and PCR identification.
  • the obtained cells of the 9 clones were extracted into the genome, and 50 ng of genomic DNA was taken for PCR detection.
  • PCR detection of the upstream primer is 3'HRF (see SEQ ID NO. 11): 5 -GGTGGGATTAGATAAATGCCTGC-3 ', designed in the sequence neo
  • the downstream primer is 3'HRR (see SEQ ID NO. 12): 5 -GAGTTGAGTAAAAAGTATTGCGG-3 ', designed to the outside of the 3' homology arm, only cells with homologous recombination can amplify the target band (Fig. 11).
  • PCR detection The results showed that one of the 9 clones obtained had homologous recombination, and a 6731 bp target fragment was amplified (Fig. 12).
  • the amplified target fragment was sequenced and the results showed that the PCR product was obtained.
  • the 5' end is the neomycin gene sequence, and the 3' end is the homologous right arm flanking sequence (Fig. 13), which is consistent with the expected sequence after homologous recombination, indicating that the clone point is indeed a positive target cell, human lysobacteria
  • the enzyme gene was integrated into the bovine asl-casein locus.
  • the target cell clone was then used for nuclear transfer.
  • Example 2 Asl-casein locus site-specific integration of other target gene targeting vectors (taking human lactoferrin gene as an example)
  • total mRNA was extracted from human breast tissue and reverse transcribed to obtain cDNA.
  • NCBI published human lactoferrin gene sequence (Genebank No.: NM_002343) to design upstream and downstream primers: hLF upstream primer, TGCAAGCTTACCATGAAACTTGTCT (see SEQ ID NO. 13); hLF downstream primer, ACGTCTAGAAGCTGGGCCATCTTCTTCG (see SEQ ID N0.14).
  • the cDNA obtained by reverse transcription was used as a template, and a 2155 bp hLF coding region was amplified by high-fidelity enzyme.
  • Hindl1 and Xbal cleavage sites were subcloned into the PCDNA3.1 vector and sequenced. Sequencing results showed 100% homology to the published sequence.
  • a 2449 bp DNA sequence containing the human lactoferrin coding cassette and bovine growth hormone gene tailing signal (bGH pA ) was amplified using the upstream primer TGCCTCGAGACCATGAAACTTGTC (see SEQ ID NO. 15) and the downstream primer GTACTCGAGTAGAGCCCCAGCTGGT (see SEQ ID N0.16).
  • the amplified sequence Xhol was digested into the Xhol site of the LPR vector of Example 1 to complete the construction of a targeting vector for integrating the human lactoferrin gene at the bovine asl-casein locus (Fig. 15).
  • the target gene can be integrated into the bovine asl-casein locus by cell screening, and the transgenic bovine which specifically expresses the exogenous gene in the mammary gland can be obtained by nuclear transfer or the like, and the method disclosed by the present invention is similarly obtained.
  • the target gene is integrated into the locus of cattle, sheep, rabbits and other animals such as asl-casein, casein, Y-casein, ⁇ -lactoglobulin, etc., which can be efficiently expressed in the mammary gland and processed by nuclear transfer.
  • a transgenic animal having a breast-specific and highly efficient expression of a foreign gene is obtained.
  • Target genes such as lysozyme, human lactoferrin gene, blood protein, therapeutic monoclonal antibody, etc. can be integrated into asl-casein, ⁇ -casein, ⁇ -casein, ⁇ -milk of animals such as cattle, sheep and rabbits.
  • Globulin can be efficiently expressed in the mammary gland at a locus that is highly efficiently expressed in the mammary gland.

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Abstract

La présente invention concerne un procédé d'une intégration spécifique au site de gènes exogènes. Les gènes exogènes sont intégrés de manière spécifique au site dans le locus des caséines as1bovines par le procédé de ciblage génique puis les gènes exogènes peuvent obtenir la région régulatrice de la caséine as1 et la région régulatrice du locus de la caséine pour accomplir une expression hautement efficace desdits gènes exogènes dans la glande mammaire bovine.
PCT/CN2011/000183 2010-11-25 2011-01-31 Procédé d'une intégration spécifique au site de gènes exogènes WO2012068769A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103589740A (zh) * 2012-08-16 2014-02-19 中国科学院上海生命科学研究院 人体内含有外源重组dna来源的转录本、dna片段、或其翻译产物，及其检测方法和应用

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106119203A (zh) * 2015-05-08 2016-11-16 内蒙古大学 TALENs介导的奶山羊成纤维细胞基因敲除和基因定点插入的方法
CN106636198A (zh) * 2016-10-31 2017-05-10 华中农业大学 猪乳腺特异高表达乳铁蛋白基因的定点敲入质粒载体及其应用
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1502694A (zh) * 2002-11-20 2004-06-09 上海杰隆生物工程股份有限公司 通过Cre-LoxP定点打靶体细胞克隆家畜作为乳腺生物反应器的研制
CN1659959A (zh) * 2004-02-24 2005-08-31 中国人民解放军军事医学科学院生物工程研究所 用核移植制备产组织型纤溶酶原激活剂突变体转基因山羊的方法
CN1914324A (zh) * 2004-11-23 2007-02-14 韩国生命工学研究院 利用同源重组的牛β－酪蛋白基因靶向载体

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1502694A (zh) * 2002-11-20 2004-06-09 上海杰隆生物工程股份有限公司 通过Cre-LoxP定点打靶体细胞克隆家畜作为乳腺生物反应器的研制
CN1659959A (zh) * 2004-02-24 2005-08-31 中国人民解放军军事医学科学院生物工程研究所 用核移植制备产组织型纤溶酶原激活剂突变体转基因山羊的方法
CN1914324A (zh) * 2004-11-23 2007-02-14 韩国生命工学研究院 利用同源重组的牛β－酪蛋白基因靶向载体

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHENG, G. ET AL.: "Study on targeting the human lysozyme gene on alphasl-casein locus for transgenic bovine production", ADVANCES IN CHINA ANIMAL GENETICS AND BREEDING - THE FIFTEENTH SESSION OF THE NATIONAL ANIMAL GENETICS AND BREEDING SYMPOSIUM, 10 October 2009 (2009-10-10), pages 117 *
KOLB, A.F. ET AL.: "Insertion of a foreign gene into the beta-casein locus by Cre-mediated site-specific recombination", GENE, vol. 227, 1999, pages 21 - 31, XP004155764, DOI: doi:10.1016/S0378-1119(98)00607-6 *
MAGA, E.A. ET AL.: "Expression of human lysozyme mRNA in the mammary gland of transgenic mice", TRANSGENIC RESEARCH, vol. 3, 1994, pages 36 - 42 *
SHEN, WEI ET AL.: "High-efficient Gene Targeting of Goat Mammary Epithelium Cell by the Multi-selection Mechanism", ACTA GENETICA SINICA, vol. 32, no. 4, April 2005 (2005-04-01), pages 366 - 371 *
YU, Z. ET AL.: "Expression and Bioactivity of Recombinant Human Lysozyme in the Milk of Transgenic Mice", J. DAIRY SCI., vol. 89, 2006, pages 2911 - 2918, XP026957126 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103589740A (zh) * 2012-08-16 2014-02-19 中国科学院上海生命科学研究院 人体内含有外源重组dna来源的转录本、dna片段、或其翻译产物，及其检测方法和应用
CN103589740B (zh) * 2012-08-16 2016-09-21 中国科学院上海生命科学研究院 人体内含有外源重组dna来源的转录本、dna片段、或其翻译产物，及其检测方法和应用

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