Preparation of doc2α and doc2β gene-deficient zebrafish mutants and construction method of zebrafish autism model

A zebrafish, alpha gene technology, applied in the field of genetic engineering, can solve the problems of complex experimental process, long experimental period, unsystematic research on pathogenesis, etc., and achieve the effect of simple construction method, accurate drug effect, and accurate drug toxicity.

Active Publication Date: 2021-11-16
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the existing technology generally establishes an autism model by knocking out a certain autism-related pathogenic gene in mice; (1) only knocking out a single gene to conduct research on the pathogenesis of autism and Not comprehensive, while using mice to establish autism models consumes a lot of money, the experiment cycle is long, the reproduction process is cumbersome, and high-throughput drug screening cannot be performed
(2) The use of heavy metal treatment or injection of drugs cannot establish a stable genetic strain genetically. There are many unknown and uncertain factors that affect the establishment of the model. The research on the pathogenesis is not systematic. Every time the research is carried out, the model must be re-modeled. The experimental process is complicated and difficult. Accompanied by unstable factors, resulting in unstable modeling, and not suitable for high-throughput drug screening
(3) At present, there are relatively few studies on the establishment of autism models in zebrafish. Existing studies only knock out a certain gene alone, not all gene families, and then study the role of a certain gene in autism. The pathogenesis is not systematic

Method used

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  • Preparation of doc2α and doc2β gene-deficient zebrafish mutants and construction method of zebrafish autism model
  • Preparation of doc2α and doc2β gene-deficient zebrafish mutants and construction method of zebrafish autism model
  • Preparation of doc2α and doc2β gene-deficient zebrafish mutants and construction method of zebrafish autism model

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1, zebrafish doc2α gene and doc2β Gene expression detection

[0065] (1) Detection by semi-quantitative PCR (semi-Q-PCR) doc2α gene and doc2β The expression of the gene during the development of the wild-type zebrafish, collected 15h, 2d, 5d, 7d, 10d zebrafish small fish, and carried out mRNA expression detection; the results showed that the expression of the gene gradually increased over time (reference figure 1 A).

[0066] (2) Detection by Q-PCR doc2α gene and doc2β The expression of genes in various tissues of wild-type zebrafish, dissect and collect the heart, brain, liver, spleen, kidney, intestine, gill, skin of wild-type adult zebrafish, put them into Trizol and extract RNA, and perform reverse transcription Follow up with Q-PCR to understand doc2α gene and doc2β The expression of the gene in zebrafish and whether it is specifically highly expressed in nerve tissue; it was found by Q-PCR that the zebrafish doc2α and doc2β Genes are hig...

Embodiment 2

[0067] Example 2, CRISPR / Cas9 gene knockout target site design

[0068] doc2α gene and doc2β The design principle and synthesis method of the target site (target) of gene sgRNA are as follows:

[0069] 1. Target design:

[0070] a. Go to the database to find the genomic DNA and CDS of the knockout gene. The target should be designed after the ATG of the last isoform, and the target should be designed in the exon region;

[0071] b. The target contains 19-23 bp bases. If the T7 promoter is used to synthesize, the 5' end should be GGA, or GGG. The 3 bases adjacent to the 3' end of the target constitute the PAM region, and the required sequence is NGG (N is arbitrary bases), target sites can be selected on the sense or antisense strand. The target should be blasted on the database to ensure a single site. Note: NGG is not the target site, do not add it during synthesis. If not synthesized with T7 primer, but synthesized with sp6, the first base is G, and the second and thi...

Embodiment 3

[0118] Embodiment 3, a kind of construction method of zebrafish autism model

[0119] A method for constructing a zebrafish autism model, comprising the following steps:

[0120] S1. respectively doc2α gene and doc2β sgRNA sequences of gene target sites and Cas9 protein were co-injected into single cells of wild-type zebrafish embryos to generate chimeric F 0 generation doc2α Gene-deficient zebrafish and doc2β Gene-deleted zebrafish;

[0121] S2. Chimera F 0 generation doc2α Gene-deficient zebrafish and doc2β Gene-deleted zebrafish were crossed with wild-type zebrafish to obtain F 1 generation doc2α Heterozygous zebrafish with gene deletion doc2α + / m and doc2β Heterozygous zebrafish with gene deletion doc2β + / m ;

[0122] S3. will get F 1 generation doc2α Heterozygous zebrafish with gene deletion doc2α + / m and get F 1 generation doc2β Heterozygous zebrafish with gene deletion doc2β + / m double gene heterozygous line doc2α + / m doc...

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Abstract

The invention relates to the field of genetic technology engineering, and specifically discloses a method for preparing a doc2α and doc2β gene-deficient zebrafish mutant and a method for constructing a zebrafish autism model. The present invention utilizes CRISPR / Cas9 technology to realize double-gene-specific knockout of zebrafish doc2α gene and doc2β gene for the first time, establishes zebrafish with deletion of doc2α and doc2β genes, and forms a stable genetic zebrafish autism model. The present invention also provides a zebrafish A method for constructing a fish autism model. The constructed zebrafish autism model can be used for high-throughput drug screening more efficiently and accurately, and can more accurately evaluate drug effects and drug toxicity.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a doc2α and doc2β Preparation of gene-deficient zebrafish mutants and construction method of zebrafish autism model. Background technique [0002] Autism Spectrum Disorder (ASD) is a developmental disorder of the central nervous system and one of the most serious neurodevelopmental disorders in children. According to the World Health Organization ICD-10 diagnostic criteria for autism, the clinical manifestations of autistic patients are mainly language barriers, social activity barriers, and limited, repetitive stereotyped behaviors, sensory system abnormalities, and some patients have obvious Specific interests, abnormal or low intelligence, about 3 / 4 of the cases have significant mental retardation and so on. Patients usually develop disease symptoms at the age of 3, and the best period of training and rehabilitation is before the age of 7. At present, many fami...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/12A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/40A01K2267/03C07K14/47C12N15/8509
Inventor 迟雅丽杨新平冯涛
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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