Construction method and application of a mutant gabrg2 transgenic zebrafish epilepsy model

A construction method, zebrafish technology, applied to other methods of inserting foreign genetic materials, using microinjection, recombinant DNA technology, etc., to achieve the effect of large-scale basic and clinical research application value

Active Publication Date: 2022-03-29
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

GABA A Receptor α1 subunit (GABRA1) knockout model, in which juvenile zebrafish exhibit epileptic phenotypes such as increased activity, EEG activity, and increased mortality, but no GABRG2 mutant epileptic zebrafish line has yet been reported

Method used

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  • Construction method and application of a mutant gabrg2 transgenic zebrafish epilepsy model
  • Construction method and application of a mutant gabrg2 transgenic zebrafish epilepsy model
  • Construction method and application of a mutant gabrg2 transgenic zebrafish epilepsy model

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Construction of GABRG2 (F343L) transgenic zebrafish epilepsy model strain.

[0035] This Example 1 provides a protocol for transferring mutant GABRG2 (F343L) into fertilized zebrafish eggs by transgenic means and constructing a zebrafish model of epilepsy. Specifically include the following steps:

[0036] The first step, the construction of the transgene expression vector: use the Pubmed database to find the human GABRG2 gene sequence, synthesize the GABRG2 open reading frame sequence, mutate the 1027th base T into C (SEQ ID No: 1), and clone it into pME- MCS plasmid vector, the 5' cloning site is EcoRI, the 3' cloning site is XbaI, construct the intermediate vector middle entryclone:pME-GABRG2 1027T>C (SEQ ID No: 3). The sequence of the HuC neuron-specific promoter region was cloned, and the 5'entry clone: ​​p5E-HuC vector (SEQ ID No: 4) was constructed. Cloning 3'entry clone: ​​p3E-polyA vector (SEQ ID No: 5). The 5'entry clone, middle entry clone and 3...

Embodiment 2

[0046] Example 2 Application of GABRG2(F343L) transgenic zebrafish epilepsy model in antiepileptic drug screening.

[0047] Select clinically commonly used antiepileptic drugs, such as sodium valproate, carbamazepine, clonazepam, levetiracetam, etc., and add them to the zebrafish culture medium at concentrations of 50 μM, 100 μM, 50 μM, and 30 mM, respectively. 24 hours after the drug, the video analysis system was used to record for 30 minutes, and the parameters were collected every minute. Drugs that have a therapeutic effect on the epilepsy phenotype of transgenic zebrafish were screened by means of swimming behavior analysis. The result is as Figure 8 As shown, the above four drugs can significantly reduce the swimming distance of transgenic zebrafish and reduce their excitability, suggesting that the above drugs all have inhibitory effects on the seizures of GABRG2(F343L) transgenic zebrafish.

Embodiment 3

[0048] Construction of embodiment 3 GABRG2 (R177G) transgenic zebrafish strain

[0049] According to the method described in Example 1, different mutant GABRG2 gene expression sequences were designed, and the mutation site was the 529th base C of the human GABRG2 gene was mutated to G (SEQ ID No: 12). Construction of GABRG2 mutant expression vector pDestTol2CG2; HuC:GABRG2 529C>G -polyA, transgenic zebrafish gene lines were obtained by microinjecting zebrafish embryos.

[0050] Phenotype analysis of GABRG2(R177G) transgenic zebrafish lines:

[0051]Zebrafish juveniles at 5dpf were selected, and the movement of wild zebrafish and mutant zebrafish lines under normal conditions was recorded using a zebrafish juvenile high-throughput behavioral screening box (Danio VisionChamber) and an animal movement trajectory tracking system (EthoVision XT 13). Trajectory, recording starts at 4:00 p.m. for a total of 24 hours (14 hours of light and 10 hours of darkness). Behavioral parameter...

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Abstract

The invention discloses a method for constructing a zebrafish model of epilepsy induced by a mutant GABRG2 transgene. Using zebrafish as a model animal, the mutant human GABRG2 gene is transferred into the zebrafish to make it a zebrafish line with an epileptic phenotype. The zebrafish line can be used to study the relationship between GABRG2 mutations and the pathogenesis of epilepsy, and to screen antiepileptic drugs.

Description

technical field [0001] The invention belongs to the technical field of animal model preparation in biology and basic medical research, and specifically relates to a transgenic zebrafish epilepsy model constructed by using an epilepsy-related gene GABRG2 mutant, and a method for constructing the epilepsy zebrafish model and its effect on epilepsy. Applications in research work on pathogenesis, drug screening, and antiepileptic drug target identification. Background technique [0002] Epilepsy is a common chronic neurological disorder characterized by the presence of spontaneous, unprovoked recurrent seizures and is one of the most common disabling neurological disorders, affecting more than 50 million people worldwide, approximately 2.4 million per year Man is diagnosed with epilepsy. The increase in the frequency, duration and severity of epileptic seizures may increase the cognitive and emotional disorders of patients, lead to the occurrence of epileptic encephalopathy, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N15/85C12N15/89A01K67/027
CPCC12N15/66C12N15/85C12N15/89A01K67/0275A01K2227/40A01K2267/0356A01K67/027A61P25/08
Inventor 张琦刘东沈丁玎陈娟柯姝安
Owner NANTONG UNIVERSITY
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