Gene knockout breeding method for tnni3k gene deletion zebrafish

A gene knockout and gene deletion technology, applied in the field of gene knockout, can solve the problems of low efficiency of targeting technology and high off-target rate

Pending Publication Date: 2019-01-25
HUNAN NORMAL UNIVERSITY
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  • Abstract
  • Description
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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

Method used

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  • Gene knockout breeding method for tnni3k gene deletion zebrafish
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  • Gene knockout breeding method for tnni3k gene deletion zebrafish

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Embodiment 1

[0098] 1) Design CRISPR / Cas9 gene knockout target sites and detection primers

[0099] Query the genomic DNA sequence of the zebrafish tnni3k gene on the National Center for Biotechnology Information (NCBI), analyze its functional domain on the website SMART (http: / / smart.embl-heidelberg.de / ), and knock it out according to CRISPR / Cas9 According to the principle, the target site of tnni3k gene is designed on the website The ZiFiT Targeter (http: / / zifit.partners.org / ZiFiT / ). The selection of targets must follow this standard: 5'-GG-(N)18-NGG-3'. The GG dinucleotide at the 5' end is a 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 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 tnni3k gene, thereby changing the expres...

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Abstract

The invention relates to the technical field of gene knockout, in particular, the invention discloses a gene knockout breeding method for tnni3k gene deletion zebrafish, which adopts CRISPR/Cas9 geneediting technology to design a suitable target site on the tnni3k gene of zebrafish, and synthesizes specific gRNA and Cas9-protein was co-injected into zebrafish cells. After 48 hours of embryo culture, the embryos were selected for genotypic analysis, which confirmed the validity of the selected sites. The invention can more efficiently and more accurately silence a specific gene in the genome of an organism, and has the advantages of simple production, low cost, and can simultaneously cut a plurality of loci on a target gene and silence any number of single genes. At that same time, zebrafish embryo interfered with the tnni3k gene appear obvious developmental abnormalities.

Description

technical field [0001] The invention relates to the field of gene knockout, and in particular discloses a method for gene knockout breeding tnni3k gene-deficient zebrafish. Background technique [0002] The tnni3k (TNNI3 interacting kinase) gene is located on chromosome 8 of zebrafish, including 25 exons and 24 introns. The full-length cDNA is 2508bp, encoding 835 amino acids. tnni3k contains 10 evolutionarily conserved functional structures At the same time, through gene differential expression profile analysis and genome association analysis, it was found that tnni3k was expressed in multiple tissues in the early human embryo, especially in the heart. [0003] The genes and signaling pathways in zebrafish and human heart development have high homology, and the tnni3k gene is relatively conservative in evolution. Studies have found that the expression level of tnni3k is particularly high in the early embryonic stage of zebrafish. Moreover, compared with other animal models...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22C12N15/89C12Q1/6888C12Q1/6806C12Q1/6869
CPCC12N9/22C12N15/89C12N15/902C12Q1/6806C12Q1/6869C12Q1/6888C12Q2535/101
Inventor 邓云刘乐欧阳诗陈湘定曾宇茜
Owner HUNAN NORMAL UNIVERSITY
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