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Method for preparing mutant CNPase zebra fish model capable of reducing cardiac functions and application

A zebrafish and mutant technology, applied in the direction of using microinjection, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems that the study of heart function has not been reported yet

Pending Publication Date: 2021-04-30
科偲(山东)创业服务有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, CNPase mutant mice have been reported, but there is no report on the animal model related to CNPase mutant zebrafish, and the study on the heart function of zebrafish has not been reported yet.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] This case mainly illustrates a CNPase mutant zebrafish model of severe heart failure with bradycardia. The microinjection method of morpholino antisense oligonucleotides (MO) was used to down-regulate the zebrafish cnpase gene, and the standard control morpholino antisense oligonucleotide (control-MO-Spl) injection group was control. The development of zebrafish embryos in each group was observed under a stereo microscope, and the number of zebrafish embryo deaths and deformities at different injection concentrations were statistically analyzed.

[0022] Table 1. Survival rate (%) of zebrafish and control embryos after high-concentration cnpase-MO knockout.

[0023] Survival rate (%) control-MO-Spl cnpase-MO-Spl 24h 100 100 48h 84.21 14.65 72h 63.16 9.48 96h 63.16 9.48 120h 63.16 9.48

Embodiment 2

[0025] To ensure the number of surviving embryos, we then reduced the amount of cnnpase-MO-Spl used to 1-2 nL. On the 4th day after the injection of cnpase-MO-Spl, it was found that zebrafish embryos had cardiac effusion and abnormal development of somites in the early stage, leading to bending of the body axis. The atria and ventricles of cnpase-MO-Spl zebrafish were relatively larger compared to control-MO. In addition, the results of cardiac function evaluation indicators showed that the heartbeat, cardiac output, fractional shortening, and ejection fraction of cnpase-MO-Spl zebrafish all decreased, revealing that after knocking down cnpase, the heart cannot pump enough oxygen-rich blood to the In other parts of the body, it is suggested that knocking down cnpase can induce spontaneous severe heart failure accompanied by bradycardia and even heart failure in zebrafish.

[0026] Table 2. Cardiac function evaluation of cnpase-MO zebrafish embryos (n=3-5)

[0027] ...

Embodiment 3

[0029] Four days after using the CRISPR / Cas9-mediated cnnpase gene editing system, we found that its phenotype was the same as that injected with cnnpase-MO-Spl. Heart effusions and abnormal development of somites appeared in early zebrafish embryos, leading to body The shaft is bent. Compared with the control group, the cnpase-MO-Spl injection group showed precordial edema, abnormal cardiac ringing, weakened cardiac beating, and ventricular hypertrophy. After using two independent knockdown / knockout methods with completely different principles, the knockdown / knockout effect (phenotype) we obtained is the same, confirming that the gene knockdown method established in this project is effective and specific of.

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Abstract

The invention relates to a method for preparing CNPase mutant zebra fish, and confirms that a mutant model can spontaneously generate bradycardia and even heart failure accompanied by severe heart failure. According to the invention, a CRISPR-Cas9 technology is combined with a microinjection technology, CNPase is specifically knocked out, a CNPase mutant F1 generation is prepared, and confirmation is performed through a Sange sequencing method; and through hybridizing and screening, a zebra fish homozygous mutant strain with heart specificity marked with green fluorescence is prepared, and it is confirmed that the CNPase homozygous mutant zebra fish can spontaneously generate cardiac hypertrophy and partially develop to heart failure through cardiology and kinetics indexes. The CNPase mutant zebra fish is prepared, an animal model is provided for researching functions of CNPase, and particularly great convenience is provided for researching related diseases of bradycardia accompanied by severe heart failure.

Description

Background technique [0001] 2',3'-Cyclic-nucleotide-3'-phosphodiesterase (2',3'-Cyclic-nucleotide 3'-phosphodiesterase, CNPase), with catalytic 2',3'-cAMP and 2',3 '- Function of cGMP degradation. In the patent application (application number) of our team, the adeno-associated virus recombinant vector was used to overexpress CNPase in rat hearts ligated from the abdominal aorta. This method has a good therapeutic effect on myocardial hypertrophy. At present, CNPase mutant mice have been reported, but there is no report on the animal model related to CNPase mutant zebrafish, and the study on the heart function of zebrafish has not been reported yet. [0002] The present invention provides, for example, a method for preparing CNPase mutant zebrafish, which can provide an animal model for functional research of CNPase in the heart. The zebrafish is small, the reproduction time is short, the breeding cost is low, and it is easy to manage; the zebrafish eggs are transparent, easy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027C12N15/85C12N15/89C12N15/55
CPCA01K67/0276A01K67/0275C12N15/8509C12N15/89C12N9/16C12Y301/04037A01K2207/05A01K2217/075A01K2227/40A01K2267/0375C12N2800/106Y02A40/81
Inventor 陈凯欣王东方谭文
Owner 科偲(山东)创业服务有限公司
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