Method for selecting and breeding ALPK2 gene-deleted zebrafish through gene knock-out

A gene knockout and gene deletion 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: 2019-04-19
HUNAN NORMAL UNIVERSITY
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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
In early 2013, a new artificial endonuclease, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9, can more ef

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  • Method for selecting and breeding ALPK2 gene-deleted zebrafish through gene knock-out
  • Method for selecting and breeding ALPK2 gene-deleted zebrafish through gene knock-out
  • Method for selecting and breeding ALPK2 gene-deleted zebrafish through gene knock-out

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Embodiment 1

[0099] 1) Design CRISPR / Cas9 gene knockout target sites and detection primers

[0100] Query the genomic DNA sequence of the zebrafish ALPK2 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 ALPK2 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 ALPK2 gene, thereby changing the expression...

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Abstract

The invention relates to the technical field of gene knock-out, in particular to a method for selecting and breeding an ALPK2 gene-deleted zebrafish through gene knock-out. The method comprises the steps of designing multiple proper targeting sites on an ALPK2 gene of the zebrafish by means of a CRISPR/Cas9 gene editing technology, and synthesizing specific gRNA and Cas9-protein in vitro; conducting microscopic injection of gRNA and Cas9-protein into a cell of the zebrafish, and after 48 hours of embryo culture, selecting embryos to carry out genotype analysis, so that the effectiveness of theselected site is confirmed. The method can more efficiently and accurately silence specific genes in the genome of an organism, manufacturing is easy, and the cost is low. Moreover, the sites on thetarget gene can be simultaneously sheared at the same time to silence any number of single genes. Additionally, development deformity of the zebrafish embryos with the ALPK2 gene is interfered.

Description

technical field [0001] The invention relates to the field of gene knockout, and in particular discloses a method for gene knockout breeding ALPK2 gene-deficient zebrafish. Background technique [0002] The ALPK2 (alpha-kinase 2) gene is located on chromosome 11 of zebrafish, including 10 exons and 9 introns. The full-length cDNA is 5234bp, encoding 1037 amino acids. [0003] The genes and signaling pathways in zebrafish and human heart development are highly homologous, and the ALPK2 gene is relatively conservative in evolution. Studies have found that the expression level of ALPK2 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 transparency. [0004] Through the CRISPR / Cas9 gene editing technology, a suitable targeting site was designed on the ALPK2 g...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22A01K67/027
CPCC12N15/907A01K67/027A01K2217/075A01K2227/40A01K2267/02C12N9/22
Inventor 邓云聂静远陈湘定陈坤刘然
Owner HUNAN NORMAL UNIVERSITY
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