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Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application of zebra fish model

A technology of arrhythmia and zebrafish, applied in the field of animal model construction, can solve the problems of lack of related disease animal models and drug screening methods

Active Publication Date: 2022-08-09
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no specific drug for the prevention and treatment of metabolic encephalopathy and cardiac arrhythmia, mainly because the genetic disease has only been discovered in recent years, and there is a lack of related diseases Animal Models and Drug Screening Methods

Method used

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  • Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application of zebra fish model
  • Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application of zebra fish model
  • Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application of zebra fish model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of tango2 mutant zebrafish model

[0034] Through the human TANGO2 gene sequence, the zebrafish tango2 gene sequence was obtained by alignment on NCBI. According to the principle of the CRISPR / Cas9 knockout method, the target gRNA sequence was designed and synthesized on the second exon of tango2: GGAGCTACTAATGTACCTGT.

[0035] Use the same amount of gRNA for pairing to obtain the double-stranded gRNA primer, use the pUC57 vector to construct an in vitro transcription template, use T7 RNA polymerase for in vitro transcription of the sgRNA, and further purify and recover to obtain the sgRNA.

[0036] The in vitro transcription system is: linearization template 2 μg, 5x transcription buffer 8 μl, 10x dithiol alcohol solution 4 μl, 10x ribonucleotide buffer 4 μl, RNase inhibitor 2 μl, T7 RNA polymerase 4 μl, DEPC supplemented to 40 μl system. The reaction was carried out at 37°C for 4 hours.

[0037] A mixed working solution of sgRNA and Cas9 mRNA ...

Embodiment 2

[0048] Example 2 Phenotypic analysis of tango2 mutant zebrafish

[0049] The wild-type zebrafish and the first-generation tango2 homozygous zebrafish were selfed separately. After collecting the fertilized eggs, the physiological characteristics of zebrafish development cycle, body shape, activity, righting response, swim bladder size, and predation ability were observed. The zebrafish heartbeat rhythm was detected on the 7th day after fertilization, and the zebrafish heartbeat frequency, movement trajectory, muscle damage, and brain pathological morphology were detected on the 12th day after fertilization to verify the effectiveness of the TANGO2 gene mutation disease drug screening model.

[0050] The specific phenotypes are as follows:

[0051] 1) Morphological observation of the disease model: With the progress of growth and development, the tango2 mutant zebrafish gradually appeared symptoms such as death and weakened ability to correct response. The zebrafish was placed...

Embodiment 3

[0056] Example 3: Application of tango2 mutant zebrafish

[0057] Use the tango2 gene mutant zebrafish constructed in Example 1 to screen drugs for the treatment of TRMEA diseases, and drugs that can improve or rescue the phenotypes of tango2 mutant zebrafish larvae such as death, arrhythmia, and morphological abnormalities are the target drugs.

[0058] The wild-type control group, the tango2 mutant control group and the tango2 mutant drug-treated group were set up. A 24-well cell plate was used, and 10 zebrafish were placed in each well and cultured at 28.5 °C. The zebrafish culture water was used to prepare a 5 mM concentration of 3-pyridinecarboxylic acid amide solution. The zebrafish embryos were administered at a final concentration of 5 μM at the age of 1 day, and the phenotypic changes of tango2 mutant zebrafish such as morphology, survival, behavior, and heartbeat were observed according to the method of Example 2, and recorded for a total of 12 days. The results sho...

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Abstract

The invention provides a zebra fish model for screening medicines for improving and treating metabolic encephalopathy and arrhythmia TRMEA diseases and a construction method of the zebra fish model. Zebra fish tango2 is used as a target gene, a genome editing technology is utilized, in-vitro synthesized tango2 specific sgRNA and Cas9mRNA are microinjected into a wild zebra fish fertilized egg, and the zebra fish model is constructed. And screening generations by using specific primers to obtain the homozygous mutant zebrafish with the tango2 gene specifically knocked out. According to the invention, the genetically stable tango2 deletion mutant zebra fish can be obtained, and the mutant can be used for evaluating the treatment effect of various drugs on TRMEA diseases by utilizing the transparency of zebra fish embryos and the symptoms similar to those of TRMEA disease patients, such as lethal, arrhythmia, muscle injury and motion retardation of juvenile zebra fish of the mutant zebra fish.

Description

technical field [0001] The invention belongs to the technical field of animal model construction, and in particular relates to a zebrafish model for drug screening and a construction method and application thereof, which can be used for screening drugs for treating metabolic encephalopathy and arrhythmia diseases. Background technique [0002] Metabolic encephalopathy and arrhythmia disease (TRMEA) is a newly discovered human genetic disease in recent years, which was first reported in 2016. The initial onset time of patients with the disease is between 2 months and 8 years old. The main clinical symptoms include metabolic syndrome, encephalopathy, arrhythmia, developmental delay, rhabdomyolysis, etc. The clinical symptoms are complex and diverse. [0003] At present, there is no specific drug for the prevention and treatment of metabolic encephalopathy and arrhythmia diseases, mainly because the genetic disease has only been discovered in recent years, and animal models and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N15/85C12N15/89C12N15/113C12N15/12A61K49/00
CPCA01K67/0276C12N15/8509C12N15/89C12N15/113C07K14/461A61K49/0008A01K2217/075A01K2217/15A01K2227/40A01K2267/0306C12N2310/20Y02A40/81
Inventor 陈才勇沈帅祺赵祯祯陈梦影
Owner ZHEJIANG UNIV
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