Method for selective breeding of Lgr6 gene deletion type zebrafish through gene knock out

Abstract

The invention relates to the technical field of gene knock out, and particularly discloses a method for selective breeding of Lgr6 gene deletion type zebrafish through gene knock out. The method comprises the steps of through a CRISPR / Cas9 gene editing technique, designing an appropriate targeting site on the Lgr6 gene of zebrafish, performing in vitro synthesis of sgRNA and Cas9-mRNA, performingmicroscopic co-injection into one cell of the zebrafish, performing embryo culturing for 60h, selecting embryos, and performing genotype analysis so as to confirm the validity of the selected site. The method can enable silent 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 silent. The Lgr6 gene expression is subjected to interference, and through a heredity means, the function are researched,so that further revealing of the entire process of the skeleton shape generation and adjusting and controlling the molecular mechanisms of processes are facilitated, and the method has important significance in understanding of medical skeleton 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 knockout breeding Lgr6 gene-deficient zebrafish. Background technique [0002] The Lgr6 (leucine rich repeat containing G protein-coupled receptor 6) gene is located on chromosome 23 of zebrafish, contains 18 exons, and has a full-length cDNA of 2889bp, encoding 962 amino acids. Analysis, etc., found that Lgr6 is expressed in multiple tissues in the early stage of human embryos, especially in bone. [0003] The genes and signaling pathways in zebrafish and human skeletal development are highly homologous, and the Lgr6 gene is relatively conservative in evolution. Studies have found that the expression level of Lgr6 is particularly high in the early embryonic stage of zebrafish. Moreover, compared with other animal models, zebrafish has the advantages of small size, easy feeding, fast development, strong reproductive ability, in vitro fertilization, in vi...

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

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