Method for breeding mir196a gene deletion type zebrafish by gene knockout

Abstract

The invention relates to the technical field of gene knockout, in particular to a method for breeding mir196a gene deletion type zebrafish by gene knockout. By a CRISPR / Cas9 gene editing technology, appropriate targeting sites are designed on a mir196a gene of zebrafish, and specific sgRNA and Cas9-mRNA synthesized in vitro are microinjected into a zebrafish cell, after 36 hours of embryo culture,embryos are selected for genotyping, and accordingly, effectiveness of the selected sites is verified. Specific genes can be knocked out in a genome of an organism more efficiently and accurately, preparation is simple, the cost is low, multiple sites on a target gene can be cut simultaneously, and any number of single genes can be silenced. By exclusion of the mir196a gene by interference, the function of the gene is studied with a genetic approach, the whole process of intestinal morphogenesis and the molecular mechanism of regulating the processes can be further revealed, and the method has great significance in understanding of intestinal disease pathology and research and development of new therapeutic schemes medically.

Description

technical field [0001] The invention relates to the field of gene knockout, and in particular discloses a method for gene knockout breeding mir196a gene-deficient zebrafish. Background technique [0002] The mir196a gene is located on the 23rd chromosome of the zebrafish. The gene is distributed in the hoxc gene family. The stem-loop region is 106bp in length. The mature mir196a is 22nt. Its sequence is 5'UAGGUAGUUUCAUGUUGUUGGG-3'. The seed sequence of the gene is AGGTAGT. Through high-throughput sequencing, it was found that mir196a was highly expressed in the intestine. [0003] The genes and signaling pathways in zebrafish and human intestinal development are highly homologous, and the sequence of the mir196a gene in humans, pigs, mice and zebrafish is exactly the same. After high-throughput sequencing analysis, it was found that mir196a in pigs Expression is particularly high in the gut. Moreover, compared with other animal models, zebrafish has the advantages of small...

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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