Construction method of CAMTA2 gene deletion type zebrafish

A construction method and zebrafish technology, applied in the field of genetic engineering, can solve the problems of low efficiency of targeting technology and high off-target rate

Pending Publication Date: 2022-02-25
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Construction method of CAMTA2 gene deletion type zebrafish
  • Construction method of CAMTA2 gene deletion type zebrafish
  • Construction method of CAMTA2 gene deletion type zebrafish

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1) Design CRISPR / Cas9 gene knockout target sites and detection primers to query the genomic DNA sequence and functional domains of the zebrafish camta2 gene on NCBI, and design a pair of camta2 gene sequences on the website according to the principle of Crispr / Cas9 gene editing target site. The selection of target sites must strictly follow the following criteria: 5'-GG-18bp-NGG-3'. The GG dinucleotide at the 5' end is part of the T7 promoter, and the design of the target site is not limited by this, but it must be ensured that the 3' end of the target site ends with NGG. The selection principle of the target site is: it must be ensured that base insertion or deletion at the target site can affect the entire structural domain of camta2, thereby changing the expression of the gene.

[0056] Target site sequence:

[0057] Target site 1: 5'-AAACACTCCAGCAGCTATTGGG-3'

[0058] RC: 3'-CCCAATAAGCTGCTGGAGTGTTT-5'

[0059] Target site 2: 5'-GGAGTGTTTGCCTCCAACCCCGG-3'

[006...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a construction method of CAMTA2 gene deletion type zebrafish. A proper targeting site is designed on the CAMTA2 gene of the zebra fish through a CRISPR / Cas9 gene editing technology, so that the gene editing efficiency is improved, and the off-target effect is reduced. The CAMTA2 gene expression in the constructed zebrafish model is changed, and the heart development is influenced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for constructing CAMTA2 gene-deleted zebrafish. Background technique [0002] Right heart failure due to right ventricular hypertrophy remains a major cause of morbidity and mortality in children with congenital heart disease. Congenital pulmonary hypertension, tetralogy of Fallot, and transposition of the great arteries (anatomical right ventricle is functional left ventricle) are the main causes of right ventricular hypertrophy in this population. The transition from RV hypertrophy to RV failure undergoes a remodeling phase characterized by structural and functional changes in cardiomyocytes that are unique to the RV. Throughout the remodeling process, a metabolic shift towards aerobic glycolysis, mitochondrial dysfunction leads to inadequate energy supply, while excitation-contraction coupling impairment and abnormal collagen deposition lead to systolic d...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/11C12N15/113C12N9/22C12N15/89A01K67/027
CPCC07K14/461C12N15/113C12N9/22C12N15/89A01K67/0276C12N2310/20A01K2217/075A01K2227/40A01K2267/03
Inventor 谢立龚克谢婷罗勇郭慧王磊
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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