A vector and recombinant cells for goat blg knockout based on talen-mediated gene targeting

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: 2018-06-19
NORTHWEST A & F UNIV +1
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

Screening for loss-of-function mutants is complicated by the random and unpredictable nature of modifications obtained by this method
In addition, the random integration of helper vectors has a great impact on the safety of individual organisms

Method used

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  • A vector and recombinant cells for goat blg knockout based on talen-mediated gene targeting
  • A vector and recombinant cells for goat blg knockout based on talen-mediated gene targeting
  • A vector and recombinant cells for goat blg knockout based on talen-mediated gene targeting

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Experimental program
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Embodiment Construction

[0038] In order to improve the gene targeting efficiency of knocking out goat β-lactoglobulin gene, the present invention uses TALEN (transcription activator-like effector nuclease) to identify the first goat β-lactoglobulin (C.hircus beta-lactoglobulin) gene A site on the exon region generates a double-strand break after cutting, and initiates DNA double-strand break repair. At the same time, a homology arm vector is introduced, and the DNA recombination repair process triggered by DNA double-strand breaks is used to make the assembled 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 with shorter homology arms, co-transfection of goat fetal fibroblasts, drug screening and identification A fur...

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Abstract

The invention discloses a vector and a recombinant cell for knocking out goat BLG based on TALEN-mediated gene targeting. According to the goat β-lactoglobulin gene, the present invention designs and constructs a TALEN eukaryotic expression vector targeting its first exon and a BLG gene knockout vector containing a shorter homology arm, and uses the TALEN expression vector as a template in vitro Transcribed mRNAs were co-transfected into goat fetal fibroblasts, and β-lactoglobulin gene knockout cells were obtained after drug screening and identification. The present invention uses TALEN technology to greatly improve gene targeting efficiency, and at the same time, the use of shorter homology arms can more conveniently and efficiently screen target cells, and the use of mRNAs avoids random integration of TALEN expression vectors in the genome.

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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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