Construction method and application of zebra fish intractable epilepsy model

A technology for refractory epilepsy and construction methods, which can be applied to other methods of inserting foreign genetic materials, using microinjection methods, using vectors to introduce foreign genetic materials, etc., and can solve problems such as no GABRG2 mutant epileptic zebrafish line reports, etc. Achieve the effect of large basic and clinical research application value

Inactive Publication Date: 2020-06-19
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

GABA A Receptor α1 subunit (GABRA1) and γ2 subunit (GABRG2) knockout ...

Method used

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  • Construction method and application of zebra fish intractable epilepsy model
  • Construction method and application of zebra fish intractable epilepsy model
  • Construction method and application of zebra fish intractable epilepsy model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of GABRG2 (P282S) transgenic zebrafish epilepsy model strain.

[0036] Example 1 provides a protocol for transferring mutant GABRG2 (P282S) into fertilized zebrafish eggs by transgenic means and constructing a zebrafish model of epilepsy. Specifically include the following steps:

[0037] The first step is 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 844th base C into T (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 entry clone: ​​pME-GABRG2 844C>T (SEQ ID No: 3). The sequence of the HuC neuron-specific promoter region was cloned, and the 5'entryclone: ​​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'e...

Embodiment 2

[0043] Example 2 Transcriptome analysis of GABRG2 (P282S) transgenic zebrafish.

[0044] The transcriptome of the GABRG2 mutant zebrafish line was analyzed, and the specific steps were as follows: Based on the Illumina sequencing platform, the transcriptome sequencing of the transgenic zebrafish brain tissue was performed, the differentially expressed genes of the mutant zebrafish line were analyzed, and GO enrichment analysis and KEEG pathway enrichment analysis.

[0045] The first step, RNA extraction and library construction: isolate 3dpf and 5dpf mutant and wild zebrafish brain tissues, use TRIzol reagent to extract the total RNA of each sample, and quantify the total RNA of each sample with NanoDrop and 1% agarose gel and identification, 1 μg of total RNA was used to construct a next-generation sequencing library. Using oligo dT magnetic beads to separate Poly(A) mRNA magnetism, random primers and ProtoScript II reverse transcriptase to synthesize first-strand cDNA, and ...

Embodiment 3

[0047] Example 3 The response of GABRG2(P282S) transgenic zebrafish epilepsy model to traditional antiepileptic drugs.

[0048] Select traditional antiepileptic drugs commonly used in clinical practice, including 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. 30 minutes after adding the drug, the video analysis system was used to record for 30 minutes, and the parameters were collected every minute. Analysis of the effects of antiepileptic drugs on the epileptic phenotype of transgenic zebrafish by swimming behavior. The result is as Figure 6 As shown, none of the above four drugs could significantly reduce the swimming distance of the transgenic zebrafish, further suggesting that GABRG2(P282S) transgenic zebrafish has the characteristics of refractory.

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Abstract

The invention discloses a construction method of a zebra fish intractable epilepsy model. A mutant human GABRG2 gene of which the 282th proline codon is mutated into serine is transferred into a zebrafish, so that the zebra fish becomes a zebra fish line with intractable epilepsy phenotype. The method provides an effective method basis for construction of the zebra fish intractable epilepsy model, also provides an important tool for epilepsy pathology research and antiepileptic drug screening research, and has great foundation and clinical research application value.

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 refractory epilepsy model constructed by utilizing epilepsy-related gene GABRG2 mutants, and the zebrafish refractory epilepsy model Construction methods and their application in research work such as pathogenesis and drug screening. Background technique [0002] Epilepsy is a chronic disease in which the sudden abnormal discharge of brain neurons leads to transient brain dysfunction. According to the latest epidemiological data in China, the overall prevalence rate of epilepsy in China is 7.0‰, which has become the second most common disease in neurology after headache. Although more than 30 anti-epileptic drugs (AEDs) are routinely used in clinical practice, nearly 30% of epilepsy patients are ineffective to drug treatment. Traditional AEDs mainly include γ-aminobutyric acid (GABA) enhanc...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/89A01K67/027
CPCA01K67/0278A01K2207/15A01K2227/40A01K2267/0356C12N15/85C12N15/89
Inventor 张琦刘东沈丁玎仇嘉颖王新
Owner NANTONG UNIVERSITY
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