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Construction method for CamkIIdeltaC gene overexpression induced ejection fraction reduction type heart failure

A technology of ejection fraction and overexpression, applied in other methods of inserting foreign genetic materials, genetic engineering, plant gene improvement, etc., can solve the problems of time-consuming, low connection efficiency, etc., achieve high efficiency, low cost, and improve transcription The effect of efficiency

Pending Publication Date: 2022-06-24
DONGFANG HOSPITAL BEIJING UNIV OF CHINESE MEDICINE
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Problems solved by technology

[0005] The purpose of the present invention is to provide a construction method for heart failure with reduced ejection fraction caused by the overexpression of the CamkIIδC gene, so as to solve the above-mentioned deficiencies in the technology, and the present invention realizes the improvement of the fault tolerance rate by using the conventional CRISPR-Cas9 in vitro transcription technology At the same time, it improves the efficiency of transcription and reduces the cost of transcription. Compared with the commonly used methods such as TA cloning, restriction enzyme cloning and blunt end cloning, the connection efficiency is low, specific restriction enzyme sites are required, and time-consuming Shortcomings such as longer, the In-Fusion Cloning method adopted in the present invention can clone any DNA fragment into any linearized vector, and fully ensure the high fidelity of the target gene after the PCR reaction

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  • Construction method for CamkIIdeltaC gene overexpression induced ejection fraction reduction type heart failure
  • Construction method for CamkIIdeltaC gene overexpression induced ejection fraction reduction type heart failure
  • Construction method for CamkIIdeltaC gene overexpression induced ejection fraction reduction type heart failure

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Embodiment

[0046] Construction method of heart failure with reduced ejection fraction caused by overexpression of CamkIIδC gene:

[0047] Query NCBI to obtain the mouse CaMKIIδB cDNA sequence, use CRISPR / Cas9 in vitro transcription technology to obtain Cas9 mRNA and gRNA, and construct a 3.6kb 5' homology arm, CAG-LSL-CaMKIIδC-3xFlag- The homologous recombination vector of IRES-tdTomato and 3.3kb 3' homology arm, by means of homologous recombination, the CAG-LSL-CaMKIIδC-3xFlag-IRES-tdTomato expression cassette was inserted into the Rosa26 gene locus, and Cas9 mRNA, The gRNA and donor vector were microinjected into the fertilized eggs of C57BL / 6J mice, and after the genotype in the tail of the mice was identified by PCR technology, the F0 generation of mice with conditional overexpression of CaMKIIδC was obtained;

[0048] Then F0 generation mice were mated with wild-type C57BL / 6J mice to obtain F1 generation mice, and F1 generation mice were mated with myocardial-specific Myh6-Cre-ERT2 ...

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Abstract

The invention discloses a construction method for CamkII delta C gene overexpression induced ejection fraction reduction type heart failure, which comprises the following steps: constructing a CaMKII delta C gene overexpression mouse by using a Crisp-cas9 transgenic technology; tamoxifen is injected every other day, CaMKII delta C gene overexpression is induced, and the heart failure phenotype with reduced ejection fraction appears in the mouse after one week. Meanwhile, by using a conventional CRISPR-Cas9 in-vitro transcription technology, the effect of improving the error-tolerant rate is achieved, meanwhile, the transcription efficiency is improved, the transcription cost is reduced, and compared with common methods such as TA cloning, restriction enzyme cutting cloning and smooth end cloning, the method has the defects that the connection efficiency is low, specific restriction enzyme cutting sites are needed, and consumed time is long; according to the invention, any DNA fragment can be cloned into any linearized vector by adopting the In-Fusion Cloning method, and after the PCR reaction is carried out, the high fidelity of the target gene is fully ensured.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a construction method for heart failure with reduced ejection fraction caused by overexpression of CamkIIδC gene. Background technique [0002] Heart failure is a complex clinical syndrome in which the initial myocardial injury of various reasons causes changes in the structure and function of the heart, resulting in impaired ventricular filling or ejection capacity. It is currently believed that the basic mechanism of the development of chronic heart failure is the Pathological remodeling, the progression of heart failure includes two main key processes, one is myocardial death, and the other is a systemic response caused by excessive activation of the neuroendocrine system, mainly related to the renin-angiotensin-aldosterone system and the sympathetic system. Intervention of these two key processes is the basis for effective prevention and treatment of heart failure. However, due t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113C12N15/55C12N15/54C12N15/90C12N15/89A01K67/027
CPCC12N15/8509C12N15/1137C12N9/22C12N15/907C12N15/89C12N9/12A01K67/0278C12Y207/11017C12N2310/20A01K2207/15A01K2207/20A01K2217/072A01K2217/15A01K2227/105A01K2267/0375
Inventor 刘静林谦万洁徐江林蔡芸
Owner DONGFANG HOSPITAL BEIJING UNIV OF CHINESE MEDICINE
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