A method for gene knockout and selection of stat1a gene-deficient zebrafish

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 and breeding of stat1a gene deletion zebrafish. Background technique [0002] The STAT1 (signal transducer and activator of transcription 1) gene is located in human 2q32.3 and encodes a transcription factor of 750 amino acids. It is generally believed that this gene mediates the interferon signaling pathway, which can directly regulate the transcription of target genes and participate in cell proliferation and differentiation. Through gene differential expression profiling and genomic association analysis, it was found that STAT1 gene is closely related to osteoporosis. [0003] The genes and signaling pathways of zebrafish and humans during skeletal development are highly homologous, and the STAT1 gene is evolutionarily conserved. The two different splices of human STAT1 gene, STAT1-alpha and STAT1-beta, correspond to the two splices of zebrafish...

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

In early 2013, a new artificial endonuclease, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) 9, can more efficiently and accurately silence specific genes in the genome of organisms, and is simple and The cost is low, and multiple sites on the target gene can be cut at the same time, and any number of single genes can be silenced, but at the same time, this technology has certain defects, and its off-target rate is relatively high

Images