Construction method and application of dilated cardiomyopathy mouse model

A dilated cardiomyopathy, mouse model technology, applied in the field of medical biology

Active Publication Date: 2021-05-25
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the function of PRMT5 in dilated cardiomyopathy has not been reported

Method used

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  • Construction method and application of dilated cardiomyopathy mouse model
  • Construction method and application of dilated cardiomyopathy mouse model
  • Construction method and application of dilated cardiomyopathy mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1. Study on the relationship between PRMT5 expression changes and dilated cardiomyopathy

[0045] Analyzing the results of human dilated cardiomyopathy (DCM) heart gene chip published in the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information, 101 DCM patient samples (including 15 samples of GSE1145) from the GEO database were analyzed. samples and 86 samples of GSE5406) ​​and 27 control samples (11 samples of GSE1145 and 16 samples of GSE5406) ​​were analyzed for PRMT5 expression, and it was found that the expression of PRMT5 was significantly reduced in the heart of human DCM patients ( figure 1 ).

Embodiment 2

[0046] Example 2, Preparation of Cardiomyocyte-specific Prmt5 Gene Knockout Mice

[0047] 1.1 Prmt5 conditional gene targeting mice (Prmt5 fl / fl mouse) construction

[0048] The PRMT5 protein of mouse is the protein shown in GenBank Accession No.NP_038796 (UpdateDate 2021-01-03) in amino acid sequence, mouse Prmt5 gene (wild-type allele, Prmt5 + ) includes 17 exons, and the genome sequence where it is located is the complementary sequence of the 54744639-54754927 nucleotide sequence of GenBank Accession No.NC_000080 (Update Date 2020-09-22), and the 54754927 nucleotide sequence is recorded as It is the first nucleotide of the Prmt5 gene, wherein the 1-224th is the first exon sequence, the 856-974th is the second exon sequence, and the 1280-1365th is the third exon Sequence, No. 2034-2168 is the fourth exon sequence, No. 2584-2696 is the fifth exon sequence, No. 2825-2874 is the sixth exon sequence, No. 4017-4180 is the seventh exon sequence Exon sequence, 5317-5478 is the e...

Embodiment 3

[0079] Example 3. Detection of Prmt5 gene knockout efficiency in cardiomyocyte-specific Prmt5 gene knockout mice

[0080] Get the Prmt5-cKO mice prepared in Example 2 and the Prmt5 fl / fl Heart tissue of mice.

[0081] (1) Protein level detection

[0082] Heart tissue protein extraction: Add 500 μL RIPA lysate (pre-added protease inhibitors and phosphatase inhibitors) to the centrifuge tube containing heart tissue, use a homogenizer to thoroughly homogenize the tissue, and wash the rotor with PBS during the process. The homogenized tissue was placed on ice for 10 min.

[0083] Place the centrifuge tube into a sonicator for sonication. Ultrasound program: Ultrasonic time 3min 30s, work 6s, interval 9s.

[0084] 12000r / min, centrifuge at 4°C for 20min.

[0085] The resulting supernatant was aspirated into another clean centrifuge tube, and the protein concentration was measured by the BCA method. The standard curve was obtained by using the measured standard concentration, ...

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Abstract

The invention discloses a construction method and application of a dilated cardiomyopathy mouse model. The dilated cardiomyopathy model mouse can be constructed by a method for specifically knocking out the Prmt5 gene in myocardial cells of the mouse. The mouse with cardiac muscle cell specificity Prmt5 deletion has pathological characteristics of dilated cardiomyopathy (DCM), including thinning of heart ventricular wall, cardiac cavity dilation, myocardial fibrosis, systolic function impairment and rising of pathological marker gene expression, which finally causes death of the mouse. The mouse DCM model constructed by the method can be applied to research on development or screening of dilated cardiomyopathy drugs.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a method for constructing a mouse model of dilated cardiomyopathy and its application. Background technique [0002] Dilated cardiomyopathy (DCM) is a complex cardiomyopathy characterized by left ventricular dilation and systolic dysfunction. Between one quarter and one half of DCM cases are thought to have a genetic basis and are classified as familial DCM, which is associated with mutations in genes encoding cytoskeletal, actin, and mitochondrial proteins. In addition, there is still a considerable part of dilated cardiomyopathy with unknown etiology, called idiopathic cardiomyopathy, and its molecular mechanism remains to be fully elucidated. [0003] Protein arginine methylation is an important post-transcriptional modification process widely existing in mammalian cells. Protein arginine methyltransferase 5 (PRMT5) is an important component of the protein arginine methy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N9/22C12N15/54A01K67/02A01K67/027A61K49/00
CPCC12N15/8509C12N9/22C12N9/1007A01K67/02A01K67/0276A61K49/0008C12Y201/01023A01K2207/15A01K2217/075A01K2227/105A01K2267/0375
Inventor 杨晓李振华王剑徐京平程萱
Owner ACADEMY OF MILITARY MEDICAL SCI
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