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Gene mapping integration and expression system and application thereof

A technique of expressing cassettes and transgenic cells, applied in genetic engineering, using microcapsules, using microinjection, etc., can solve problems such as uncertain expression of exogenous genes

Active Publication Date: 2017-07-25
SHANGHAI TRANSGENIC RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide and establish a high-efficiency transgenic sheep positional integration expression system to solve the problem of uncertain expression of foreign genes caused by uncertain integration sites and integrated copy numbers of transgenes, so that the expression of transgenes becomes controllable and predictable

Method used

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  • Gene mapping integration and expression system and application thereof
  • Gene mapping integration and expression system and application thereof
  • Gene mapping integration and expression system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Construction of Homologous Recombination Expression Vector pTG6-DsRed Containing Loxp Sequence

[0129] Design the G6 site gene targeting vector, use the red fluorescent protein DsRed as the target gene, puromycin as the positive selection gene, and diphtheria toxin as the negative selection gene to construct a plasmid, and connect two Loxp sites containing different core sequences on both sides of the target gene Point (Lox2272, Lox66), using homologous recombination to realize the anchoring of the Loxp sequence at the G6 site.

[0130] Specifically, pKO-V913 was used as the backbone vector, and the negative selection gene diphtheria toxin (Diphtheria Toxin A, DT) was inserted into the multiple cloning site by RsrII single enzyme digestion to obtain pKO V913-DT.

[0131] Using the plasmid pDsRed-N1 as a template, use Lox2272-containing primer F1: CCGCTCGAGATAACTTCGTATAGGATACTTTATACGAAGTTATTAGTTATTAATAGTAATCAAT (SEQ ID NO.:5), and primer R1: GAATGATATCGATCTGACGGTTCACTAA...

Embodiment 2

[0137] Construction and activity verification of Talen left and right arm expression vectors

[0138] A complete TALEN sequence includes a binding region on the left, a cleavage region, and a binding region on the right. Referring to the gene sequence of the G6 site, according to the principle of TALEN target site design, select two 14-19bp sequences separated by 17-18bp as the target site, and design three left arms and two right arms respectively. The specific sequences are as shown in the figure below Show:

[0139]L1: GCTCCTGATAAATTAGTTC (SEQ ID NO.: 15);

[0140] L2: GCTCCTGATAAATTAGTT (SEQ ID NO.: 16);

[0141] L3: GCTCCTGATAAATTAGT (SEQ ID NO.: 17);

[0142] R1: TGCTTGCGTTGAGGTAT (SEQ ID NO.: 18);

[0143] R2: GCTTGCGTTGAGGTATT (SEQ ID NO.: 19).

[0144] Corresponding to the above-mentioned target recognition domains respectively, the Talen backbone vector ( figure 2 ) and a gene cloning kit for gene cloning, and constructed five Talen eukaryotic expression vecto...

Embodiment 3

[0148] Preparation of cell lines expressing red fluorescent protein anchored by Loxp

[0149] Isolate goat fetal fibroblast cell line by tissue block method, culture it in a medium containing 2mM glutamine, 1mM sodium pyruvate, 1× non-essential amino acid, 2ng / mL basic fibroblast growth factor, 1000units / mL Mouse leukemia inhibitory factor, 10% fetal bovine serum (GIBCO), 100units / mL of penicillin and streptomycin in Glasgow essential medium (GMEM, GIBCO), the cells were stored at 37 ° C, 5% CO 2 Cultured in an incubator, after the second passage, the cells in the logarithmic growth phase were taken to prepare for gene transfection.

[0150] Figure 5 A schematic diagram of homologous recombination at the G6 site of the homologous recombination vector pTG6-DsRed is shown. The constructed targeting vector pTG6-DsRed was linearized by SalI, and 15 μg of the linearized vector DNA fragment was recovered, mixed with 7 μg of TALEN left and right arm plasmids (pTL1, pTR2) and co-tr...

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Abstract

The invention relates to a gene mapping integration and expression system and application thereof. Particularly, the invention relates to integration and high-efficiency expression of a nucleic acid constructor at a specific site in a transgenic sheep genome, wherein the nucleic acid constructor contains a homologous arm sequence combined with a chromosome region, a site-specific recombination sequence, an exogenous gene expression cassette and a selection marker gene expression cassette. By delivering the constructor into a host cell, mapping integration and expression of a target gene in a host cell genome can be achieved, a transgenic cell can be obtained, and transgenic sheep expressing the target gene can be obtained with the transgenic cell as a donor cell. By means of the site-specific recombination technique, expression of other specific exogenous genes and genetic modification of a transgene can be achieved in somatic cells of the transgenic sheep.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene positioning integrated expression system prepared by genetic engineering technology and its application. Background technique [0002] The exogenous protein expressed by transgenic animals has precise post-transcriptional processing and post-translational modification (glycosylation, disulfide bond formation, etc.), and the expression product is closer to the natural protein in terms of molecular structure, physical and chemical properties and biological functions. When preparing some recombinant proteins with complex post-translational modifications, the recombinant proteins produced by transgenic mammalian expression systems often have better activity. However, compared with other expression systems, the high expression of recombinant proteins in transgenic animals has problems such as long development period and high comprehensive cost, which is mainly due to the existence ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/85C12N15/87C12N15/88C12N15/89C12N15/90A01K67/027
CPCA01K67/0275A01K2227/102A01K2267/01C12N15/8509C12N15/87C12N15/88C12N15/89C12N15/907C12N2800/107C12N2810/40C12N2999/007
Inventor 成国祥俞慧清陈建泉陆平
Owner SHANGHAI TRANSGENIC RES CENT
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