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Method for preparing non-human mammal with iGb3S gene knockout and applications

A non-human mammal, gene knockout technology, applied in animal delivery, veterinary instruments, and the use of vectors to introduce foreign genetic material, etc., can solve the problem of not being able to obtain model animals

Active Publication Date: 2014-10-29
NAT INST FOR FOOD & DRUG CONTROL
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Problems solved by technology

[0008] However, some of the above studies are limited to the scope of personal laboratory research, and there is no iGb3S gene knockout mouse with independent intellectual property rights in China
However, due to the existence of international technical barriers, relevant model animals cannot be obtained in China.

Method used

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  • Method for preparing non-human mammal with iGb3S gene knockout and applications

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Embodiment 1

[0074] 1. Construction of targeting vector

[0075] 1.1 Genomic DNA was isolated from normal C57BL / 6J mice, and PCR was used to amplify the 5' end C1 (586bp), A (547bp) and 3' end B (486bp), C2 (585bp) fragments of exon 5 respectively , A and B fragments are located at the 5' end and 3' end of the catalytic functional region of exon 5, respectively, and C1 and C2 fragments are respectively located at the 5' end of A fragment and the 3' end of B fragment.

[0076] Schematic diagram of the target site figure 1 Shown: the gene sequence is selected from the genome DNA of chromosome 4 of C57BL / 6J mouse (GRCm38.p1 C57BL / 6J, NCBI Reference Sequence: NC_000070.6). The knockout gene part is located in exon 5, and its length is 687bp in the full length of the catalytic region of exon 5 plus 255bp at the 5' (upstream) end, and the total length is 942bp. The length of the targeting vector used in the present invention is 25.2kb ( figure 1 -A), containing 5' (upstream) homology arm (8...

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Abstract

The invention relates to the field of preparing gene knockout animal models, and particularly relates to a method for preparing a non-human mammal with iGb3S gene knockout and applications. The method includes: separating an iGb3S gene from genome DNA, amplifying through a long-chain RCR method to obtain a homologous arm, compositing with an antibiotic resistance gene to construct a targeting vector, transferring the targeting vector into an embryonic cell, injecting the recombinant embryonic cell into a surrogate animal embryo, transplanting the embryo into a pseudopregnancy animal, mating with an normal animal, subjecting the obtained chimera animals to genotype verification, screening the chimera animal with positive gene knockout and success gene knockout, further mating with wild type animals to obtain F1 heterozygotes, mating the F1 heterozygotes to obtain a homozygote animal with both two chromosomes being deleted, and establishing to obtain a gene knockout animal population.

Description

technical field [0001] The present invention relates to the field of making gene knockout animal models, in particular to a method and application for preparing iGb3S gene knockout non-human mammals. Background technique [0002] Biogenic materials composed of mammalian extracellular matrix are widely used in surgical wound repair, tissue reconstruction, and tissue engineering scaffold materials due to their good biocompatibility. Xenogeneic organs have also become one of the potential ways to solve the serious shortage of donor organs in organ transplantation. However, the immunological problems brought about by the application of animal-derived biological materials or xenogeneic organ tissues to the human body directly affect the safety and effectiveness of the use of such materials. The biggest obstacle to xenotransplantation is the hyperacute immune rejection (Hyperacute rejection, HAR), which occurs within a few days after xenotransplantation. From minutes to several ...

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Application Information

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IPC IPC(8): C12N15/85A61D19/04A61D19/00A01K67/027C12N5/10
Inventor 徐丽明邵安良范昌发单永强陆艳章娜杨昭鹏林永亮徐斌
Owner NAT INST FOR FOOD & DRUG CONTROL
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