Method for breeding rmnd5b (required for meiotic nuclear division 5homolog B) gene deletion type zebra fish through gene knockout

Abstract

The invention discloses a method for breeding rmnd5b (required for meiotic nuclear division 5homolog B) gene deletion type zebra fish through gene knockout and belongs to the field of gene knockout. According to the method, construction of a gRNA expression vector and gRNA in-vitro synthesis are performed through design of a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) gene knockout target site, micro-injection is performed on an embryo of the zebra fish, the effectiveness of the target site is detected, tail cutting identification is performed, TA cloning is performed on a target sequence, plasmids are subjected to Sanger sequencing, an F1 generation of heritable zebra fish mutants is obtained, the same mutant female fish and male fish are picked from mutants of the F1 generation, hybridization is performed, an F2 generation of the zebra fish mutants is obtained, F2 generation homozygote is picked from the F2 generation of the zebra fishmutants, F3 generation pure-line inheritance is performed, and an rmnd5b gene deletion type zebra fish strain is obtained. The method is lower in off-target rate and has good medical research value inresearch of the correlation between rmnd5b gene deletion and development of other organs.

Description

technical field [0001] The invention belongs to the field of gene knockout, and more specifically relates to a method for gene knockout breeding rmnd5b gene-deficient zebrafish. Background technique [0002] RMND5B (required for meiotic nuclear division 5homolog B) gene is located in human 5q35.3, encoding 391 amino acids. It is generally believed that the gene is expressed in multiple tissues of early human embryos, and the expression level is stronger in the heart, liver and kidney. Through gene differential expression profile analysis and genome association analysis, it was found that RMND5B gene is closely related to early heart development. [0003] Zebrafish and humans have high homology in genes and signaling pathways during heart development, and the RMND5B gene is relatively conservative in evolution. The human RMND5B gene corresponds to the zebrafish rmnd5b gene. Studies have found that the expression of rmnd5b is particularly high in the early zebrafish embryo ....

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problem of relatively high off-target rate existing in existing gene targeting technology, the present invention provides a method for gene knockout and selection of rmnd5b gene-deleted zebrafish, which can more efficiently and accurately silence a specific gene in the genome of an organi

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