Method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout

Abstract

A method for breeding stat1a (signal transducer and activator of transcription 1) gene-deleted zebra fish through gene knockout comprises steps as follows: design of a CRISPR / Cas9 gene knockout target site: a gRNA expression carrier is established and gRNA is synthesized in vitro; micro-injection of a zebra fish embryo; detection of effectiveness of the target site with a T7E1 method through Sanger sequencing; tail cutting identification according to the identification steps after two months of injection; TA cloning of a target sequence; Sanger sequencing of plasmids; obtaining of heritable F1 generation of a zebra fish mutant; obtaining of F2 generation homozygote of the zebra fish mutant, F3 generation pure line inheritance of the gene-deleted zebra fish with the above method, and obtaining of a new zebra fish strain.

Description

technical field [0001] The invention belongs to the technical field of gene knockout, and relates to a method for gene knockout breeding stat1a gene-deficient zebrafish. Background technique [0002] STAT1 (signaltransducerandactivatoroftranscription1) gene is located in human 2q32.3, which encodes a transcription factor of 750 amino acids. It is generally believed that this gene mediates the interferon signaling pathway, can directly regulate the transcription of target genes, and participate in processes such as cell proliferation and differentiation. Through gene differential expression profile analysis and genome association analysis, it was found that STAT1 gene was closely related to osteoporosis. [0003] The genes and signaling pathways in the skeletal development process between zebrafish and humans are highly homologous, and the STAT1 gene is relatively conservative in evolution. The two different splice forms STAT1-alpha and STAT1-beta of the human STAT1 gene cor...

AI Technical Summary

Problems solved by technology

Traditional gene targeting technology is based on embryonic stem cells (ESC) and homologous recombination technology, so the efficiency of targeting technology is extremely low

At the beginning of 2013, a new artificial endonuclease clusteredregularlyinterspacedshortpalindromicrepeats (CRISPR) / CRISPR-associated (Cas) 9, which can more efficiently and accurately silence specific genes in the genome of organisms, is simple to make, low in cost, and can be Simultaneously cut multiple sites on the target gene to silence any number of single genes, but at the same time, this technology has certain defects, and its off-target rate is relatively high

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