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Construction method and application of animal model of dilated cardiomyopathy with cardiorenal syndrome

A technology for dilated cardiomyopathy and cardio-renal syndrome, which is applied in the field of medical experimental model construction and can solve problems such as incomplete response to the pathogenesis of dilated cardiomyopathy, pathophysiological and hemodynamic changes, neuroendocrine activation, and poor model stability. , to achieve the effect of high molding rate and stable model

Pending Publication Date: 2021-12-24
THE FIRST HOSPITAL OF HEBEI MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a construction method and application of an animal model of dilated cardiomyopathy with cardiorenal syndrome, so as to solve the problem of the poor stability of the dilated cardiomyopathy with cardiorenal syndrome model in the prior art, which fails to fully reflect the pathogenesis of dilated cardiomyopathy , pathophysiology, hemodynamic changes, neuroendocrine activation and other related processes

Method used

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  • Construction method and application of animal model of dilated cardiomyopathy with cardiorenal syndrome
  • Construction method and application of animal model of dilated cardiomyopathy with cardiorenal syndrome
  • Construction method and application of animal model of dilated cardiomyopathy with cardiorenal syndrome

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Embodiment 1

[0025] The specific steps of the method for establishing an animal model of dilated heart disease with cardiorenal syndrome of the present invention are as follows:

[0026] (1) Model construction: analyze the Npr1 gene structure, and delete exon1 (including 5'UTR) of the gene. Two sgRNAs were designed in the upstream ~ 2.6kb region of ATG and downstream of exon1 respectively, resulting in about 4.0kb genome deletion and achieving the purpose of Npr1 gene knockout. Model mice were prepared using the EGE system developed by Biocytogen based on CRISPR / Cas9. Program such as figure 1 -A shown.

[0027] (2) Target sequence sequencing: Different strains may have different target gene sequences. In order to ensure the efficiency of the designed Cas9 / sgRNA, it is first necessary to perform PCR amplification and sequencing verification on the C57BL / 6N mouse tail target site sequence to ensure that the sgRNA recognition sequence is completely consistent with the C57BL / 6N mouse tail D...

Embodiment 2

[0053] Example 2 Model Evaluation

[0054] Raw materials and excipients: 8-10 weeks old male C57BL / 6N mice and male Npr1+ / - mice, distilled water. Main equipment: small animal ultrasound imaging system Vevo3100, LabChart data acquisition and analysis software system, HE staining kit, Bio-Rad TGX Stain-Free acrylamide gel-free kit, agarose gel electrophoresis instrument, Bio-Rad Bio-Rad electrophoresis instrument, Invitrogen gel imaging system, ODYSSEY two-color infrared laser imaging system, optical microscope.

[0055] Obtain F1 generation mouse by the construction method of embodiment 1, carry out genotype identification, Npr1 + / - Mice up to 8-10 weeks old were used for the following experiments. The remaining mice continued to be routinely raised in the IVC system. Npr1 + / - Male mice were compared with C57BL / 6N wild male mice, and the changes in mouse weight, heart rate, blood pressure, cardiac function, and cardiac shape were observed to evaluate the model.

[0056] P...

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Abstract

The invention provides a construction method and application of an animal model of dilated cardiomyopathy with a cardiorenal syndrome. The animal model of the dilated cardiomyopathy with the cardiorenal syndrome is constructed in a mode of specifically knocking out an animal Npr1 gene. The specific knockout of the animal Npr1 gene is carried out by utilizing a CRISPR / Cas technology, and the construction method comprises the following steps of: (a) target sequence sequencing: carrying out PCR amplification and sequencing verification on a target site sequence of the animal; (b) designing a pair of corresponding sgRNA sequences according to the target sequence of the Npr1 gene ; and (c) micro-injecting Cas9 / sgRNA into fertilized eggs of the animal, culturing to obtain an F0-generation animal, mating the positive F0-generation animal with a wild animal to obtain F1-generation animal with a stable genotype, and detecting the genotype of the F1-generation animal. Based on the Npr1 <+ / -> mouse, the animal model of dilated cardiomyopathy with the cardiorenal syndrome is constructed, model construction is simple, a model forming rate is high, and an operation is easy.

Description

technical field [0001] The invention relates to the technical field of medical experiment model construction, in particular to a construction method and application of an animal model of dilated cardiomyopathy with cardiorenal syndrome. Background technique [0002] Dilated cardiomyopathy (dilated heart disease) has seriously threatened the safety and health of human beings, and its main manifestations are enlargement of ventricular cavity and hyposystolic function of left ventricle. Symptoms are not obvious in the early stage until symptoms and signs of heart failure such as dyspnea, shortness of breath, and edema of both lower extremities appear. In the late stage, severe arrhythmias may occur, such as sinoatrial block, third-degree atrioventricular block, and ventricular fibrillation. Become one of the causes of death and pose a huge threat to life. With the progressive aggravation of cardiac insufficiency, progressive aggravation of renal insufficiency may occur, causin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113C12N15/89A01K67/027A61K49/00
CPCC12N15/8509C12N15/113C12N15/89A01K67/0276A61K49/0008C12N2310/20A01K2207/15A01K2217/075A01K2227/105A01K2267/0375A01K2267/035
Inventor 黄耀孟李曈昕刘睿思刘超
Owner THE FIRST HOSPITAL OF HEBEI MEDICAL UNIV
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