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Method for obtaining transgenic bovine fetal fibroblast by using Cas9 cutting nuclease-mediated Ipr1 fixed point insertion

A technology of ipr1 and gene, applied to cells modified by introducing foreign genetic material, genetic engineering, plant gene improvement, etc., can solve the problems of limited application of gene insertion, achieve the effect of reducing cytotoxicity and improving safety

Inactive Publication Date: 2018-01-23
NORTHWEST A & F UNIV
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Problems solved by technology

However, the current application of CRISPR / Cas9 technology-mediated gene insertion in transgenic livestock is very limited

Method used

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  • Method for obtaining transgenic bovine fetal fibroblast by using Cas9 cutting nuclease-mediated Ipr1 fixed point insertion
  • Method for obtaining transgenic bovine fetal fibroblast by using Cas9 cutting nuclease-mediated Ipr1 fixed point insertion
  • Method for obtaining transgenic bovine fetal fibroblast by using Cas9 cutting nuclease-mediated Ipr1 fixed point insertion

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Embodiment Construction

[0037] The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments. What has been described is by way of explanation, not limitation, of the invention.

[0038] After proving that the insertion of the mouse Ipr1 gene can limit the proliferation of Mycobacterium bovis by in vitro challenge experiments, the present invention firstly constructed the macrophage-specific targeting vector pIpr1-eGFP-P2A-Puro containing the Ipr1 gene, and then The targeting vector pIpr1-eGFP-P2A-Puro and the CRISPR / Cas9 expression vector targeting the targeting site were co-transfected into bovine fetal fibroblasts by electroporation. After puromycin drug screening, the targeting vector was identified by PCR and Southern Blotting. positive clones. The targeted positive cloned cells were used as nuclear donors to transfer bovine enucleated oocytes to obtain transgenic cloned embryos. Finally, the transgenic cloned cattle are obtained ...

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Abstract

The invention discloses a method for obtaining transgenic bovine fetal fibroblast by using Cas9 cutting nuclease-mediated Ipr1 fixed point insertion. The bovine fetal fibroblast is cotransfected by adonor vector and a CRISPR / Cas0 expression vector for a target locus through an electroporation method, an MSR1 promoter in the donor vector can realize specific expression in full-time phagocytic cells, and after puromycin drug screening, the targeted positive clone cell is obtained through PCR authentication. The transgenic positive clone cell is used as a donor cell so as to obtain a transgenicclone embryo, the transgenic clone embryo is further transplanted into the uterus of a rutting acceptor calf, and finally a living Ipr1 fixed point insertion transgenic cloned calf is obtained.

Description

technical field [0001] The invention belongs to the technical field of transgenic cloning animals, relates to the construction of nuclear donor cells of transgenic cloned cattle, and particularly relates to a method for obtaining targeted bovine fetal fibroblasts by using Cas9 cutting nuclease to mediate the fixed-point insertion of Ipr1 gene. Background technique [0002] Tuberculosis is a worldwide zoonotic disease caused by the transmission of Mycobacterium tuberculosis (MTB) between humans and cattle, which poses a serious threat to the production safety of animal husbandry, the safety of animal products and even human health. [0003] The Ipr1 (intracellular pathogen resistance 1, intracellular pathogen resistance factor 1) gene is a gene that has been found to mediate the natural immunity of Mycobacterium tuberculosis, which can limit the reproduction of Mycobacterium tuberculosis in macrophages and can Regulates the death pattern of macrophages (Behar, 2011). The dis...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC07K14/35C12N15/65C12N15/85C12N15/907C12N2510/00C12Q1/66
Inventor 张涌袁梦珂高元鹏韩静马晓楠吴腾
Owner NORTHWEST A & F UNIV
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