Gene knockout method for breeding selection of fhl1b gene-deletion type zebra fish

Abstract

The invention relates to the technical field of gene knockout and particularly discloses a gene knockout method for breeding selection of a fhl1b gene-deletion type zebra fish. According to the method, by means of a CRISPR / Cas9 gene editing technique, an appropriate target locus is designed on a fhl1b gene of the zebra fish, specific sgRNA and Cas9-mRNA which are synthesized outside the fish bodyare microscopically injected into one cell of the zebra fish, and 60 hours after embryo cultivation is carried out, by selecting embryos to conduct genotype analysis, the effectiveness of the selectedlocus is proved. The off-target rate is low, not only is the fhl1b gene removed by interference, but also more convenience is provided for further disclosing the whole process of generation of a heart shape and regulating and controlling a molecular mechanism of the process by adopting a genetic approach to studying the functions of the fhl1b gene, and the method has a significant meaning in understanding of medical pathology of heart diseases and research and development on new therapeutic schemes.

Description

technical field [0001] The invention relates to the field of gene knockout, and in particular discloses a method for gene knockout and selection of fhl1b gene-deficient zebrafish. Background technique [0002] The fhl1b (four and a half LIM domains 1b) gene is located on chromosome 10 of zebrafish and contains 6 exons and 5 introns. The full-length cDNA is 1512bp, encoding 280 amino acids. The study found that using the Morpholino interference technology to interfere with the fhl1b gene in zebrafish embryos, obvious developmental deformities appeared. At the same time, through gene differential expression profile analysis and genome association analysis, it was found that fhl1b was present in multiple tissues in the early stages of human embryos. expressed in the heart, especially in the heart. [0003] The genes and signaling pathways in zebrafish and human heart development are highly homologous, and the fhl1b gene is relatively conservative in evolution. Studies have fou...

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