Method of preparing alpha-1,3GT gene knockout non-human mammal and application

A non-human mammal, gene knockout technology, applied in the direction of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of not being able to obtain model animals

Inactive Publication Date: 2015-07-22
NAT INST FOR FOOD & DRUG CONTROL
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  • Abstract
  • Description
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Problems solved by technology

[0008] However, some of the above-mentioned studies are limited to the scope of personal laboratory research, and there is no α-1,3GT 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 of preparing alpha-1,3GT gene knockout non-human mammal and application
  • Method of preparing alpha-1,3GT gene knockout non-human mammal and application
  • Method of preparing alpha-1,3GT gene knockout non-human mammal and application

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Embodiment 1

[0060] 1. Construction of targeting vector

[0061] 1.1 Genomic DNA was isolated from normal C57BL / 6J mice, and PCR was used to amplify the 5' end C1 (582bp), A (558bp) and 3' end B (525bp), C2 (559bp) 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.

[0062] Schematic diagram of the target site figure 1 Shown: the gene sequence is selected from the genomic DNA of chromosome 2 of C57BL / 6J mouse (GRCm38.p1C57BL / 6J, NCBI Reference Sequence: NC_000068.7). The knockout gene part is located in exon 9, and its length is 694bp in the full length of the catalytic region of exon 9 plus 246bp at the 5' (upstream) end, and the total length is 940bp. The length of the targeting vector used in the present invention is 18.966kb ( figure 1 ), containing 5' (upstream) homology arm (4...

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Abstract

The invention relates to the field of manufacturing a gene knockout animal model, in particular to a method of preparing an alpha-1,3GT gene knockout non-human mammal and an application. The preparation method comprises the steps of separating an alpha-1,3GT gene from genomic DNA (Deoxyribose Nucleic Acid), carrying out amplification via a long-chain PCR (Polymerase Chain Reaction) method to obtain a homologous arm, compositing with an antibiotic drug resistance gene to construct a targeting vector, transferring the targeting vector into an embryonic cell, injecting the recombined embryonic cell into an embryo of a surrogacy animal, transplanting into a pseudopregnancy animal body for mating with a normal animal, carrying out genotype verification on an obtained chimera animal, screening a positive gene knockout chimera animal with successful gene knockout for further mating with a wild animal to obtain F1 generation heterozygotes, obtaining a homozygote animal with two chromosomes removed after mating of the F1 generation heterozygotes, and establishing a line 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 α-1,3GT 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 severa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/54C12N5/10A01K67/027G01N33/53
Inventor 徐丽明邵安良范昌发陆艳单永强章娜杨昭鹏徐斌张伟
Owner NAT INST FOR FOOD & DRUG CONTROL
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