Method for knocking out zebrafish slc26a4 gene

A zebrafish and gene technology, applied in the field of gene knockout, can solve the problems of high cost, many steps, time-consuming, reagent and sequencing cost, etc., and achieve the effect of simple production and low cost

Inactive Publication Date: 2020-05-15
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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Problems solved by technology

[0005] However, the current general method for detecting CRISPR/Cas9 mutations in zebrafish gene knockout has certain defects. For example, it takes a lot of time and reagents to cut the tail of zebrafish in the F0 generation, and do TA cloning and San...

Method used

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  • Method for knocking out zebrafish slc26a4 gene
  • Method for knocking out zebrafish slc26a4 gene
  • Method for knocking out zebrafish slc26a4 gene

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Embodiment 1

[0043]1) Design CRISPR / Cas9 gene knockout target sites and detection primers

[0044] Query the genomic DNA sequence of the zebrafish slc26a4 gene on the National Center for Biotechnology Information (NCBI), analyze its functional domain on the website SMART (http: / / smart.embl-heidelberg.de / ), and knock out it according to CRISPR / Cas According to the principle, the target site of the slc26a4 gene was 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 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 slc26a4 gene, thereby changing the ex...

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Abstract

The invention relates to the technical field of gene knockout, in particular to a method for knocking out a zebrafish slc26a4 gene. Through a CRISPR/Cas9 gene editing technology, a proper targeting site is designed on a zebrafish slc26a4 gene, and specific sgRNA and Cas9-mRNA are synthesized in vitro for gene editing of zebra fish. The invention can more efficiently and more accurately silence specific genes, is simple to manufacture and low in cost, can simultaneously shear multiple sites on a target gene and silence any number of single genes, and also provides a method for breeding slc26a4gene deletion type zebrafish through gene knockout, a zebrafish model is constructed by the method. The method is helpful for further revealing the whole process of human large vestibular aqueduct syndrome and a molecular mechanism for regulating the process, and has very important significance in understanding the pathology of vestibular aqueduct and researching and developing a new treatment scheme in medicine.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a method for knocking out the zebrafish slc26a4 gene. Background technique [0002] The slc26a4 gene is located on chromosome 4 of zebrafish, including 20 exons and 19 introns. The full-length cDNA is 2390bp, encoding 760 amino acids. slc26a4 encodes an anion transport membrane protein, namely pendrin protein. Pendrin protein is a member of the anion transport family slc26, which mainly mediates the transport of Cl-, HCO3-, OH-, I- and formate. SLC26A4 gene mutation is associated with Pendred syndrome (PDS) (dilated vestibular aqueduct or with inner ear Teratogenic deafness and goiter) are closely related to large vestibular aqueduct syndrome (EVA), and it was found that slc26a4 was expressed in multiple tissues in early human embryos, especially in the inner ear. [0003] The genes and signaling pathways in the development of zebrafish and human inner ear are highly homol...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N15/89A01K67/027A01K67/02
CPCA01K67/02A01K67/0276A01K2207/15A01K2217/075A01K2227/40A01K2267/03C12N15/113C12N15/89C12N15/902C12N2310/20
Inventor 谢鼎华赖若沙谢华平付贵芳杨曙曾婷谢缤灵邓慧玲胡昱瑶
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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