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Construction method and application of a mouse model of dilated cardiomyopathy

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

Active Publication Date: 2022-07-05
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 a mouse model of dilated cardiomyopathy
  • Construction method and application of a mouse model of dilated cardiomyopathy
  • Construction method and application of a mouse model of dilated cardiomyopathy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

Embodiment 2

[0046] Example 2. Preparation of cardiomyocyte-specific Prmt5 knockout mice

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

[0048] The mouse PRMT5 protein is the protein whose amino acid sequence is GenBank Accession No.NP_038796 (UpdateDate 2021-01-03), the mouse Prmt5 gene (wild-type allele, Prmt5 + ) includes 17 exons, the genomic sequence of which 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 is recorded It is the first nucleotide of the Prmt5 gene, wherein the 1-224th position is the first exon sequence, the 856-974th position is the second exon sequence, and the 1280-1365th position is the third exon sequence Sequence, the 2034-2168 position is the fourth exon sequence, the 2584-2696 position is the fifth exon sequence, the 2825-2874 position is the sixth exon sequence, and the 4017-4180 position is the seventh exon ...

Embodiment 3

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

[0080] Take the Prmt5-cKO mice and Prmt5 prepared in Example 2 fl / fl Mouse heart tissue.

[0081] (1) Detection of protein level

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

[0083] Put the centrifuge tube into the sonicator for sonication. Ultrasound program: Ultrasound time 3min 30s, working 6s, interval 9s.

[0084] 12000r / min, 4 ℃ centrifuge centrifuge 20min.

[0085] The resulting supernatant was pipetted 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 method for constructing a mouse model of dilated cardiomyopathy and its application. The dilated cardiomyopathy model mouse can be constructed by specifically knocking out the Prmt5 gene in mouse cardiomyocytes. The cardiomyocyte-specific Prmt5-deficient mice constructed by the present invention have pathological features of dilated cardiomyopathy (DCM), including cardiac ventricular wall thinning, cardiac cavity dilation, myocardial fibrosis, impaired contractile function, and pathological marker genes Expression rises, eventually leading to the death of the mice. The mouse DCM model constructed by the method of the present invention can be applied to the research of developing or screening 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. One-quarter to 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 widespread in mammalian cells. Protein arginine methyltransferase 5 (PRMT5) is an important component of the protein arginine methyltransferase f...

Claims

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Application Information

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Patent Type & Authority Patents(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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