Screening kit for malignant arrhythmia, establishment method and application of animal model

An arrhythmia, animal model technology, applied in the field of biomedical disease diagnosis

Active Publication Date: 2018-09-28
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] However, there is no report on the relationship between the A690E mutation site in the RYR2 gene and sudden cardiac death.

Method used

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  • Screening kit for malignant arrhythmia, establishment method and application of animal model
  • Screening kit for malignant arrhythmia, establishment method and application of animal model
  • Screening kit for malignant arrhythmia, establishment method and application of animal model

Examples

Experimental program
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Effect test

Embodiment 1

[0042] The patient was a 56-year-old female who was admitted to the Department of Cardiology, Shanghai Tenth People's Hospital. Electrocardiogram showed typical ventricular tachycardia. Investigation of the patient's family medical history revealed that his grandfather and grandmother were consanguineous marriages, and his father died of heart disease in his early years. The specific cause of death was not recorded due to the long history. The patient had three sisters. The patient was the elder sister. The third sister died suddenly when she was 45 years old while chasing a bus on the way to work. The patient's son was 19 years old, a freshman in college, and died suddenly while playing basketball. Based on the clinical electrocardiographic pattern and family genetic history, we highly suspected that he had CPVT. According to the recommendations of the American Heart Association guidelines, the patients underwent genetic testing. We took the patient's peripheral blood, ext...

Embodiment 2

[0044] We established an animal model to verify whether the mutation is a CPVT pathogenic mutation.

[0045] We knocked the mutation site into the mouse RYR2 gene using CRISPR / Cas9 technology. The main method is as follows:

[0046] 1. Cas9mRNA using mMESSAGE T7Ultra Kit for in vitro transcription; GuideRNA using MEGAshortscript TM Kit was transcribed in vitro, and the products were all used MEGAclear TM Kit purification.

[0047] 2. The injection sample was prepared into a 100 μl system, the sample was injected into the male pronucleus of fertilized eggs, and the donor fertilized eggs were taken from C57BL / 6J.

[0048] 3. F 0 On the 14th day after birth, 0.3 cm of the mouse tail was clipped, and 400 μl of lysate and 40 μl of proteinase K (10 mg / ml) were added, and digested overnight at 56°C. The supernatant was collected by centrifugation, precipitated with twice the volume of anhydrous ethanol, washed with 75% ethanol, dried slightly, dissolved in 100 μl of pure wate...

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PUM

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Abstract

The invention provides a screening kit for malignant arrhythmia, an establishing method and an application of the animal model. In the method one RyR2 mutation site (p. A690E) is detected in patient families with familial sudden cardiac death history and confirmed. A transgenic animal model is further applied, and a gene knock-in mouse is successfully built. The phenotype of sudden death and ventricular tachycardia induced by the mutation is verified by the mouse model, and the effects of mutation on the structure and function of calcium channel are further investigated by biochemistry, electrophysiology, cell and structural biology, disclosing the molecular mechanism of catecholaminergic polymorphic ventricular tachycardia (CPVT) induced by mutation. The method provides a new idea for thetreatment of CPVT and the prevention of sudden death, and can be applied to the mouse model to screen the arrhythmia treatment drugs.

Description

technical field [0001] The invention relates to the technical field of biomedical disease diagnosis, in particular to the identification and detection of the RYR2 gene mutation site that induces malignant arrhythmia, the establishment of an animal model and its application in drug screening. Background technique [0002] Sudden cardiac death (sudden cardiac death, SCD) refers to sudden death due to cardiac causes, which can occur in patients with known heart disease or in healthy people with no history of heart disease. Sudden cardiac death is often manifested by sudden loss of consciousness. Death usually occurs within 1 hour of the onset of acute symptoms, but a small number of survivors of cardiac arrest may die beyond this time limit. Sudden cardiac death is the most common cause of sudden natural death in developed countries. In the United States, cardiovascular disease-related deaths account for about one-third of the total death population, and sudden cardiac death ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/705C12Q1/6883C12M1/00C12M1/34A01K67/027
CPCA01K67/0275A01K2217/07A01K2227/10A01K2227/105A01K2227/107A01K2267/0375C07K14/705C12Q1/6883C12Q2600/156
Inventor 刘铮徐亚伟唐恺张敬莹强苏静熊婧钱云云郭荣李海玲苏杨刘伟静
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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