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Preparation of doc2alpha and doc2beta gene deleted zebrafish mutant and construction method of zebrafish autism model

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

Active Publication Date: 2021-06-22
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] However, in the prior art, an autism model is generally established 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. 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 single gene, 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 doc2alpha and doc2beta gene deleted zebrafish mutant and construction method of zebrafish autism model
  • Preparation of doc2alpha and doc2beta gene deleted zebrafish mutant and construction method of zebrafish autism model
  • Preparation of doc2alpha and doc2beta gene deleted zebrafish mutant and construction method of zebrafish autism model

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Experimental program
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Effect test

Embodiment 1

[0064] Embodiment 1, zebrafish doc2α gene and doc2β gene expression situation detection (1) Utilize semi-quantitative PCR (semi-Q-PCR) to detect the expression situation of doc2α gene and doc2β gene in wild-type zebrafish development process, collect 15h, 2d, 5d, 7d, and 10d zebrafish small fish were tested for mRNA expression; the results showed that the expression level of the gene gradually increased over time (reference figure 1 A). (2) Use Q-PCR to detect the expression of doc2α gene and doc2β gene in various tissues of wild-type zebrafish, dissect and collect the heart, brain, liver, spleen, kidney, intestine, gills, skin of wild-type adult zebrafish, Put it into Trizol and extract RNA, perform reverse transcription and then perform Q-PCR to understand the expression of doc2α gene and doc2β gene in zebrafish and whether they are specifically highly expressed in nerve tissue; through Q-PCR detection, it was found that, Zebrafish doc2α and doc2β genes are highly expressed...

Embodiment 2

[0065] Example 2, CRISPR / Cas9 gene knockout target site design

[0066] The design principle and synthesis method of the target site (target) of doc2α gene and doc2β gene sgRNA are as follows:

[0067] 1. Target design:

[0068] a. Go to the database to find the genomic DNA and CDS of the knockout gene, and design the target after the ATG of the last isoform, and the target is designed in the exon region;

[0069] b.target contains 19-23bp bases, if the T7 promoter is used to synthesize, the 5' end should be GGA, or GGG, and 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 third bases are purine ...

Embodiment 3

[0105] Embodiment 3, a kind of construction method of zebrafish autism model

[0106] A method for constructing a zebrafish autism model, comprising the following steps:

[0107] S1. Co-inject the sgRNA sequences of the target sites of the doc2α gene and the doc2β gene and the Cas9 protein into single cells of wild-type zebrafish embryos to obtain chimeric F 0 Generation doc2α gene-deleted zebrafish and doc2β gene-deleted zebrafish;

[0108] S2. Chimera F 0 The doc2α gene-deleted zebrafish and the doc2β gene-deleted zebrafish were crossed with wild-type zebrafish respectively to obtain F 1 Generation of heterozygous zebrafish doc2α with deletion of the doc2α gene + / m Heterozygous zebrafish doc2β with deletion of the doc2β gene+ / m ;

[0109] S3. will get F 1 Generation of heterozygous zebrafish doc2α with deletion of the doc2α gene + / m and get F 1 Generation of heterozygous zebrafish doc2β with deletion of the doc2β gene + / m Doc2α was obtained by crossing th...

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Abstract

The invention relates to the field of gene technology engineering, and particularly discloses preparation of a doc2alpha and doc2beta gene deleted zebrafish mutant and a construction method of a zebrafish autism model. According to the invention, double-gene specific knockout of the doc2alpha gene and the doc2beta gene of the zebrafish is realized for the first time by utilizing a CRISPR / Cas9 technology, the doc2alpha and doc2beta gene deleted zebrafish is established, and the zebrafish autism model with stable inheritance is formed. The invention also provides the construction method of the zebrafish autism model. The built zebrafish autism model can be used for high-throughput drug screening more efficiently and accurately, and the drug effect and drug toxicity can be evaluated more accurately.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing a doc2α and doc2β gene-deficient zebrafish mutant and a method for constructing a 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, ...

Claims

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Application Information

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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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