Method for breeding tcf25 gene deletion type zebra fish through gene knockout

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

Inactive Publication Date: 2018-05-04
HUNAN NORMAL UNIVERSITY
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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...

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  • Method for breeding tcf25 gene deletion type zebra fish through gene knockout
  • Method for breeding tcf25 gene deletion type zebra fish through gene knockout
  • Method for breeding tcf25 gene deletion type zebra fish through gene knockout

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Embodiment 1

[0079] 1) Design CRISPR / Cas9 gene knockout target sites and detection primers

[0080] On the National Center for Biotechnology Information (NCBI), query the genomic DNA sequence of the zebrafish tcf25 gene, on the website SMART ( http: / / smart.embl-heidelberg.de / ) to analyze its functional domains, according to the principle of CRISPR / Cas knockout, on the website The ZiFiT Targeter ( http: / / zifit.partners.org / ZiFiT / ) to design the target site of the tcf25 gene. The selection of targets must follow this standard: 5'-GG-(N)18-NGG-3'. 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 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 tcf25 gene, thereby changing the expression of ...

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Abstract

The invention relates to the technical field of gene knockout, and particularly discloses a method for breeding a tcf25 gene deletion type zebra fish through gene knockout. The method comprises the steps of through a CRISPR/Cas9 gene editing technology, designing an appropriate targeting gene locus on a tcf25 gene of the zebra fish, synthesizing in vitro to obtain specific sgRNA and Cas9-mRNA, microinjecting into a cell of the zebra fish, and after culturing an embryo for 60h, selecting the embryo for carrying out genotyping, so that the effectiveness of the selected locus is verified. The method provided by the invention is lower in off-target rate, removes the tcf25 gene through interference, is beneficial to further revealing the whole process of cardiac morphogenesis and a molecular mechanism regulating the process through researching functions through a genetics means, and is of great importance on understanding a cardiac disease pathology and researching and developing a new therapeutic schedule medically.

Description

technical field [0001] The invention relates to the field of gene knockout, and in particular discloses a method for gene knockout breeding tcf25 gene-deficient zebrafish. Background technique [0002] The tcf25 (transcription factor 25) gene is located on chromosome 18 of zebrafish, including 18 exons and 17 introns. The full-length cDNA is 1920bp, encoding 645 amino acids. tcf25 contains 3 evolutionarily conserved functional domains , the study found that using the Morpholino interference technology to interfere with the tcf25 gene in zebrafish embryos, obvious developmental deformities appeared. At the same time, through gene differential expression profile analysis and genome association analysis, it was found that tcf25 was expressed in multiple tissues in the early stages of human embryos , especially strongly expressed in the heart. [0003] The genes and signaling pathways in zebrafish and human heart development have high homology, and the tcf25 gene is relatively ...

Claims

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Application Information

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IPC IPC(8): C12N15/89C12N15/90A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/40A01K2267/0375C07K14/461C12N9/22C12N15/89C12N15/902
Inventor 邓云廖艳伟吴秀山郭芬李玲玉
Owner HUNAN NORMAL UNIVERSITY
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