Method for breeding zebra fish with wnt16 gene deletion through gene knockout

A gene knockout and gene deletion technology, applied in genetic engineering, biochemical equipment and methods, animal husbandry, etc., can solve the problems of low efficiency and high off-target rate of targeting technology

Inactive Publication Date: 2016-12-07
HUNAN NORMAL UNIVERSITY
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  • Abstract
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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
At the beginning of 2013, a new artificial endonuclease clustered regularly interspacedshortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) 9

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  • Method for breeding zebra fish with wnt16 gene deletion through gene knockout
  • Method for breeding zebra fish with wnt16 gene deletion through gene knockout
  • Method for breeding zebra fish with wnt16 gene deletion through gene knockout

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Embodiment Construction

[0083] The present invention will be further described below in conjunction with accompanying drawing:

[0084] Include the following steps:

[0085] Step 1: CRISPR / Cas9 gene knockout target site design

[0086] Query the genomic DNA sequence and functional domain of the zebrafish wnt16 gene on the GenBankTM, Ensembl or UCSC databases, and design a pair of target sites for the wnt16 gene according to the principle of CRISPR / Cas knockout. The selection of the target sites must follow the 5'- 20bp-NGG-3' standard: the GG dinucleotide at the 5' end is part of the T7 promoter, and there is no such restriction when designing the target site, but it must be ensured that the 3' end of the target site is NGG, and the target site The selection must ensure that the insertion or deletion of bases at the target site can affect the entire domain of the wnt16 gene, thereby altering gene expression,

[0087] Two pairs of specific PCR primers are as follows:

[0088] F1 (target site a forw...

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Abstract

The invention discloses a method for breeding zebra fish with wnt16 gene deletion through gene knockout. Compared with the prior art, the method has the advantages that specific genes can be more efficiently and more accurately silenced in a genome of a living body, in addition, the preparation is simple, the cost is low, moreover, a plurality of sites on a target gene can be simultaneously shorn, and single genes of any number can be silenced. The method is used for carrying out the relevant researches on gene and skeletal development, also can be used for carrying out the exploration research of other aspects, is used for detecting that whether the deletion of the wnt16 gene has relevance with the development of other organs, such as the heart, or not, and has a very good medical research value, meanwhile, the zebra fish with the wnt16 gene knocked out has the obviously shortened growth cycle, and thus the commercial value is good.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a method for gene knockout breeding wnt16 gene-deficient zebrafish. Background technique [0002] The Wnt gene is located at human 12q13 and encodes a transcription factor of 356 amino acids. It is generally believed that the Wnt signal transduction pathway is divided into canonical Wnt signaling pathway and non-canonical Wnt signaling pathway. The canonical Wnt signaling pathway is also known as the Wnt / p-catenin signaling pathway. The molecular mechanism of Wnt / p-catenin signal transduction is highly conserved in different species. Wnt16 is a non-canonical wnt ligand. It is generally believed that this gene mediates the β-catenin signaling pathway, can negatively regulate RANK signaling, inhibit the formation of osteoclasts, and participate in processes such as bone formation and bone resorption. Through gene differential expression profile analysis and genome associat...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
CPCC12N15/8509A01K67/0276A01K2217/075A01K2227/40A01K2267/02A01K2267/03C07K14/461C12N2800/106C12N2800/80C12N2810/10
Inventor 陈湘定刘薇薇苏幸邵梦思邓云谭丽君邓红文
Owner HUNAN NORMAL UNIVERSITY
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