A method for gene knockout and breeding of stat1a gene-deficient zebrafish

Abstract

The invention provides statla gene deletion type zebra fish. After an experiment design region is knocked out, the sequence is represented as SEQ ID No.1; an experiment comprises the following steps: designing a CRISPR / Cas9 gene knockout target point: constructing a gRNA expression vector and synthesizing gRNA in vitro; carrying out micro-injection of zebra fish embryos; detecting the effectiveness of the target point by a T7E1 method and Sanger sequencing; two months after injection, carrying out tail shearing and identification and carrying out identification steps above; carrying out TA cloning of a target sequence; carrying out Sanger sequencing of plasmids; obtaining an F1 generation of descendible zebra fish mutants; obtaining F2 generation homozygotes of the zebra fish mutants; and carrying out F3 generation homozygous heredity of the gene deletion type zebra fish according to the method above to obtain a new zebra fish line.

Description

technical field [0001] The invention belongs to the technical field of gene knockout, and relates to a stat1a gene-deleted zebrafish. Background technique [0002] STAT1 (signal transducer and activator of transcription 1) gene is located in human 2q32.3, which encodes a transcription factor of 750 amino acids. It is generally believed that this gene mediates the interferon signaling pathway, can directly regulate the transcription of target genes, and participate in processes such as cell proliferation and differentiation. Through gene differential expression profile analysis and genome association analysis, it was found that STAT1 gene was closely related to osteoporosis. [0003] The genes and signaling pathways in the skeletal development process between zebrafish and humans are highly homologous, and the STAT1 gene is relatively conservative in evolution. The two different splice forms STAT1-alpha and STAT1-beta of the human STAT1 gene correspond to the two Different ...

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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