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Method for obtaining transgenic bovine fetal fibroblast by use of single Cas9 nickase for mediation of fixed point insertion of NRAMP1

A FSCN1, gene technology, applied in the field of bovine fetal fibroblasts, can solve the problem of limited application of gene insertion, and achieve the effect of reducing cytotoxicity and improving safety

Active Publication Date: 2017-04-26
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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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

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  • Method for obtaining transgenic bovine fetal fibroblast by use of single Cas9 nickase for mediation of fixed point insertion of NRAMP1
  • Method for obtaining transgenic bovine fetal fibroblast by use of single Cas9 nickase for mediation of fixed point insertion of NRAMP1
  • Method for obtaining transgenic bovine fetal fibroblast by use of single Cas9 nickase for mediation of fixed point insertion of NRAMP1

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Embodiment Construction

[0036] 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.

[0037] The present invention first constructs the macrophage-specific targeting vector pNRAMP1-eGFP-P2A-Puro containing the NRAMP1 gene, and then electroporates the targeting vector pNRAMP1-eGFP-P2A-Puro and a single CRISPR targeting site / Cas9 nickase expression vectors were co-transfected into bovine fetal fibroblasts, and after puromycin drug selection, the positive clone cells for targeting were identified by PCR and Southern Blotting. 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 by transplanting the transgenic cloned embryos into the uterus of the recipient cow in estrus.

[0038] The specific reagents and materi...

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Abstract

The invention discloses a method for obtaining a transgenic bovine fetal fibroblast by use of single Cas9 nickase for mediation of fixed point insertion of NRAMP1, according to the method, an electroporation method is used for co-transfecting a bovine fetal fibroblast with a donor vector and a single CRISPR / Cas9 nickase expression vector aiming at a targeting site, a NRAMP1 gene self promoter in the donor vector enables specific expression of the NRAMP1 in a professional phagocyte, and after puromycin drug screening, a targeting positive clone cell can be obtained by PCR and Southern Blotting identification. A transgenic cloned embryo can be obtained from the transgenic positive clone cell as a nuclear donor, and a survival NRAMP1-fixed-point-inserted transgenic cloned cow can be finally obtained by further transplantation of the transgenic cloned embryo into a uterus of a rutting receptor cow.

Description

technical field [0001] The invention belongs to the technical field of transgenic cloning animals, and relates to the construction of nuclear donor cells of transgenic cloned cattle, in particular to a method for obtaining targeted bovine fetal fibroblasts by using a single Cas9 nickase to mediate the fixed-point insertion of NRAMP1 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] NRAMP1 (natural resistance-associated macrophage protein-1), also known as SLC11A1 (solute carrier family 11A member 1), is the first gene associated with tuberculosis susceptibility discovered by researchers (Vidal S et al, 1995). When Mycobacterium tuberculosis invades macrophages, NRAMP1 promotes NO production and other pro-inflammatory resp...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A01K67/027
CPCA01K67/0276A01K2227/101A01K2267/02C07K14/47C12N5/0656C12N15/85C12N2800/107C12N2800/80
Inventor 张涌高元鹏陈琳琳刘鑫吴海波袁梦珂
Owner NORTHWEST A & F UNIV