Targeted exogenous gene integration method

An exogenous gene, site-specific integration technology, applied in the field of genetic engineering, can solve the problems of inability to achieve results, the complexity of gene expression regulation, and reduce the controllability and safety of transgenes, so as to eliminate safety concerns and avoid target gene expression. Silence, the effect of increasing the success rate

Active Publication Date: 2011-06-01
北京科芙兰德生物科学有限责任公司
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to increase the expression of exogenous genes and overcome the influence of the position effect on the transgene, people have tried a variety of methods, such as adding nuclear matrix attachment elements (MAR), site control elements (LCR), insulators ( Insulator) and other elements to overcome the influence of position eff...

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: as1-casein locus site-directed integration of other target gene targeting vector construction (taking human lysozyme gene as an example)

[0032] 1. Construction of gene targeting vector

[0033] 1.1 Amplification and sequencing of homology arms

[0034] According to the bovine as1-casein sequence (Genebank number: X59856) published by GeneBank, an upstream primer (see SEQ ID NO.1) 5'-ATTT was designed GCGGCCGC TAATGTTCCTGTCATACAACTGTGAAT-3', introduce NotI restriction site; downstream primer (see SEQ ID NO.2) 5'-CC CTCGAG GTCAAGATCTATGTAAGAAAATAAAATAG-3', introduce XhoI restriction site, used to amplify homologous left arm (Left arm, 9354-11939, 2586bp). Simultaneously designed the upstream primer (see SEQ ID NO.3) 5'-ACGC GTC GAC AACCATGAAACTTCTCATCCTTAC-3', introduce SalI restriction site; downstream primer (see SEQ ID NO.4) 5'-ACGC GTCG AC ACATTCTTGGGTATGATAGCACTGC-3' introduced a SalI restriction site to amplify the homologous right arm (Right ...

Embodiment 2

[0058] Example 2: Site-directed integration of as1-casein locus and construction of targeting vectors for other target genes (taking human lactoferrin gene as an example)

[0059] According to the above vector construction method, total mRNA was extracted from human breast tissue, and cDNA was obtained by reverse transcription. The upstream and downstream primers were designed according to the human lactoferrin gene sequence published by NCBI (Genebank number: NM_002343): hLF upstream primer, TGC AAGCTT ACCATGAAACTTGTCT (see SEQ ID NO.13); hLF downstream primer, ACG TCTAGA AGCTGGGCCATCTTCTTCG (see SEQ ID NO. 14). Using the cDNA obtained by reverse transcription as a template, the 2155bp hLF coding region was amplified with high-fidelity enzymes, subcloned into the PCDNA3.1 vector using HindIII and XbaI restriction sites, and sequenced. Sequencing results showed 100% homology with the published sequence. Utilizing upstream primer TGC CTCGAG ACCATGAAACTTGTC (see SEQ ID NO....

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Abstract

The invention discloses a targeted exogenous gene integration method. Taking human lysozyme as an example, a human lysozyme gene is targetedly integrated to a bovine as1-casein loca by a gene targeting method so that a lysozyme gene captures a complete as1-casein regulatory sequence and the regulatory sequence on the casein loca; therefore, the overexpression of the human lysozyme in a bovine mammary gland is realized. By using the new method, a target gene can be targetedly integrated to a loca which contributes to self-expression; various shortcomings in conventional exogenous gene random integration in transgene are overcome; and the success rate of transgenic breeding is improved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for site-specific integration of foreign genes. Background technique [0002] 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. It is also widely used in light industry and food industry. In 2009, the U.S. FDA approved the world's first anti-thrombin transgenic drug produced by mammary gland bioreactor, which marked that the use of transgenic animals to produce recombinant proteins has truly entered the stage of industrialization. [0003] The use of mammary gland bioreactors to produce recombinant proteins has broad market application prospects, but the traditional transgenic technology still has some shortcomings in the research of mammary gland bioreactors. In traditional transgenic r...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/63C12P21/02
CPCA01K2227/101A01K2267/01A01K2217/206C12N15/8509A01K67/0278A01K2217/072
Inventor 成功汤波李宁
Owner 北京科芙兰德生物科学有限责任公司
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