Method for breeding adgrf3b gene deletion zebra fish by gene knockout

A gene knockout and gene deletion technology, applied in the field of gene knockout, can solve the problem of high off-target rate

Pending Publication Date: 2019-07-30
HUNAN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] A brand-new artificial endonuclease clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, proposed in early 2013, can more efficiently and accurately silence specific genes in the genome of organisms

Method used

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  • Method for breeding adgrf3b gene deletion zebra fish by gene knockout
  • Method for breeding adgrf3b gene deletion zebra fish by gene knockout
  • Method for breeding adgrf3b gene deletion zebra fish by gene knockout

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Embodiment 1

[0079] The method for gene knockout breeding adgrf3b gene deletion type zebrafish comprises the following steps:

[0080] Step 1: CRISPR / Cas9 gene knockout target site design and PCR detection primers

[0081] The genomic DNA sequence of the zebrafish adgrf3b gene was queried on the NCBI database, the functional domain was analyzed on the SMART ((http: / / smart.embl-heidelberg.de / ) website, and the ZiFiT Targeter ((http: / / zifit. partners.org / ZiFiT / ) to design the target site of the zebrafish adgrf3b gene, which is located on the tenth exon of the genome;

[0082] The GG dinucleotide at the 5' end is part of the T7 promoter, and this restriction is not required when designing the target site, but it must be ensured that the 3' end of the target site is NGG. The selected position of the target point must be within the structural domain of the gene to ensure that the insertion or deletion of bases at the target site can affect the entire structural domain of the adgrf3b gene, ther...

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Abstract

The invention provides a method for breeding an adgrf3b gene deletion zebra fish by gene knockout. The method comprises the following steps of: designing a proper targeting site on the adgrf3b gene ofthe zebra fish by using a CRISPR/Cas9 gene editing technology; enabling specific sgRNA and Cas9-mRNA synthesized in vitro to enter a zebra fish cell by micro co-injection; selecting embryos to carryout genotyping after carrying out embryo culture for 36 hours, so that the effectiveness of the selected site is verified. The method can knock out the specific genes in a genome of a organism more efficiently and accurately, has a simple manufacture process and low cost, and can simultaneously shear a plurality of sites on a target gene and silence any number of single genes; the adgrf3b gene isinterfered, and the function of the adgrf3b gene is researched by genetic means, so that it is beneficial to further revealing the whole morphogenesis process of intestinal tract and epithelial cellsthereof, regulating and controlling the molecular mechanism of the process, and having very important significance in the understanding of pathology of intestinal tract related diseases and the research and development of a new therapeutic scheme.

Description

technical field [0001] The invention relates to the field of gene knockout, in particular to a method for gene knockout breeding adgrf3b gene-deficient zebrafish. Background technique [0002] The adgrf3b gene is located on chromosome 20 of the zebrafish, including 14 exons and 13 introns. The full-length cDNA is 5421bp, encoding 1016 amino acids. It is found that adgrf3b is expressed in many tissues in the early stages of human embryos, especially Strongly expressed in the gut. [0003] The genes and signaling pathways in zebrafish and human intestinal development are highly homologous, and the adgrf3b gene is relatively conservative in evolution. Studies have found that the expression of adgrf3b 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 vitro development of embryos and transpa...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/90A01K67/027
CPCC07K14/461C12N15/902A01K67/0276A01K2217/075A01K2227/40
Inventor 谢华平付贵芳谢缤灵曾婷王飞英印遇龙
Owner HUNAN NORMAL UNIVERSITY
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