Method for selective breeding of ppm1g gene mutation zebrafish through gene editing

Abstract

The invention relates to the technical field of gene knock out, and particularly discloses a method for selective breeding of ppm1g gene mutation zebrafish through gene editing. The method comprises the steps of through a CRISPR/Cas9 gene editing technique, designing an appropriate targeting site on the ppm1g gene of zebrafish, performing in vitro synthesis of sgRNA and Cas9-mRNA, performing microscopic co-injection into one cell of the zebrafish, performing embryo culturing for 50h, selecting embryos, and performing genotype analysis so as to confirm the validity of the selected site. The method can enable specific genes to be efficiently and accurately mutated in organism genomes. The method is simple to make and low in cost, shearing of a plurality of sites on the target gene can be realized at the same time, and any number of individual genes can be mutated. The ppm1g gene expression is subjected to interference, and through a heredity means, the functions are researched, so that further revealing of the entire process of the heart development and adjusting and controlling the molecular mechanisms of processes are facilitated, and the method has important significance in understanding of medical heart disease pathology and research and development of a new treatment scheme.

Description

technical field [0001] The invention relates to the field of gene knockout, and in particular discloses a method for gene editing and breeding ppm1g mutant zebrafish. Background technique [0002] The ppm1g (protein phosphatase, Mg2+ / Mn2+ dependent, 1G) gene is located on chromosome 13 of zebrafish, contains 10 exons, and the full-length cDNA is 1488bp. At the same time, the ppm1g gene was found through gene differential expression profile analysis and genome association analysis. It is expressed in multiple tissues in the early stage of human embryos, especially, this gene is closely related to the neural development of zebrafish. [0003] The genes and signaling pathways of zebrafish and human heart development are highly homologous, and the ppm1g gene is relatively conservative in evolution. Studies have found that ppm1g is highly expressed in the early embryonic stage of zebrafish. Moreover, compared with other animal models, zebrafish has the advantages of small size, ...

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 clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) 9, can more efficiently and accurately mutate specific genes in the genome of organisms, and the production is simple, 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 mutated, but at the same time, this technology has certain defects, and its off-target rate is relatively high

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