TALEN-mediated vector for knocking out goat BLG through gene targeting and recombinant cell

A technology of gene targeting and gene knockout, which is applied in the field of animal genetic engineering, can solve problems such as the impact on the safety of individual organisms and complex workload, and achieve the effect of improving the efficiency of gene targeting

Active Publication Date: 2015-06-24
NORTHWEST A & F UNIV +1
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Problems solved by technology

Screening for loss-of-function mutants is complicated by the random and unpredictable nature of modifications obtained by

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  • TALEN-mediated vector for knocking out goat BLG through gene targeting and recombinant cell
  • TALEN-mediated vector for knocking out goat BLG through gene targeting and recombinant cell
  • TALEN-mediated vector for knocking out goat BLG through gene targeting and recombinant cell

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[0038] In order to improve the gene targeting efficiency of knocking out goat β-lactoglobulin gene, the present invention is mediated by TALEN (transcription activator-like effector nuclease) to recognize goat β-lactoglobulin (C.hircus beta-lactoglobulin) gene. A site on an exon region produces a double-strand break after cleavage, which initiates DNA double-strand break repair, and at the same time introduces a homology arm vector to use the DNA recombination repair process initiated by the DNA double-strand break to assemble the target DNA sequence The goat β-lactoglobulin gene is specifically inserted to realize the modification and knockout of the goat β-lactoglobulin gene. The following are the construction of TALEN eukaryotic expression vector, TALEN expression vector as template for in vitro transcription of mRNAs, construction of BLG gene knockout vector containing shorter homology arms, and co-transfection of goat fetal fibroblasts, drug screening and identification Fo...

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Abstract

The invention discloses a TALEN-mediated vector for knocking out goat BLG through gene targeting and a recombinant cell. A TALEN eukaryotic expression vector aiming at a first exon of a goat beta-lactoglobulin gene and a BLG gene knockout vector containing short homologous arms are designed and constructed according to the goat beta-lactoglobulin gene and are jointly transfect goat fetal fibroblast together with mRNAs obtained by in vitro transcription based on TALEN expression vector as a template, and a BLG gene knockout cell is obtained after drug screening and identification. According to the invention, the gene targeting efficiency is greatly improved by utilizing a TALEN technology; meanwhile, the utilization of the short homologous arms facilitates more convenient and more efficient screening of the targeted cells; random integration of the TALEN expression vector in genomes is avoided by utilizing mRNAs.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and relates to a vector and a recombinant cell for knocking out goat BLG based on TALEN-mediated gene targeting. Background technique [0002] β-lactoglobulin (BLG) is the main whey protein in ruminant milk, about 56%-60%, basically absent in human and rodent milk (Kontopidis G et al.2004), is One of the main allergens causing milk allergy in infants and young children. At present, there are many biochemical methods to reduce the allergenicity of BLG, such as heat treatment, enzymatic hydrolysis, glycosylation, and fermentation. Although these methods have a certain effect on reducing BLG allergy, they cannot fundamentally solve the sensitization problem of BLG. With the development of molecular biology technology, RNA interference technology can effectively reduce BLG in milk, and its interference efficiency can reach 100%. However, the use of RANi to produce transgenic ani...

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Application Information

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IPC IPC(8): C12N15/85C12N15/55C12N15/12C12N5/10C12N15/873
Inventor 张涌靳亚平崔趁趁刘军葛恒涛宋玉洁
Owner NORTHWEST A & F UNIV
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