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Method for building special microRNA knock-down mouse model of heart

A mouse model, a specific technology, applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, animal husbandry, etc., can solve the problems of not being able to meet the requirements, restricting embryos, and short action time

Active Publication Date: 2011-03-16
HARBIN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, its application has obvious disadvantages: high cost, repeated injections are required, among which, the more prominent ones are the short action time and the inability to be passed down
This obviously cannot meet the requirements of some disease research
The inability to be passed on to the next generation restricts some studies on embryonic development

Method used

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  • Method for building special microRNA knock-down mouse model of heart
  • Method for building special microRNA knock-down mouse model of heart
  • Method for building special microRNA knock-down mouse model of heart

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0030] 1. Construction of pBluescript-S-miR328 plasmid

[0031] The mouse mature-miR328 sequence on miRBase was selected, and its antisense sequence was designed according to the principle of base complementarity. Six copies of this antisense sequence were concatenated, each copy connected by "aatt". The sequence was digested with SalⅠ and HindⅢ, and ligated into the pBluescript vector containing the cardiac-specific promoter alph-MHC promoter. The ligation product was transformed into competent cells DH5α, and 7 colonies were picked the next morning for sequencing identification. Sequencing results showed that all seven plasmids were consistent with expectations. The recombinant plasmid pBluescript-S-miR328 was successfully established.

[0032] 2. Establishment of miR-328 transgenic mice

[0033] The above-mentioned recombinant plasmid was digested with SpeI, and the purified transgenic construct for microinjection was recovered by electrophoresis gel. This building blo...

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Abstract

The invention relates to the technical field of biotechnology, in particular to a method for building a special microRNA knock-down mouse model of a heart. The invention provides a transgenic mouse built by using a miRNA sponge technology. Exogenous gene components comprise a myocardium special promoter, a miR-328 sponge and a transcription termination signal. The myocardium miR-328 of the mouse is knocked down by almost 50%, and the mouse model is suitable for selection of antiarrhythmic drugs because the miR-328 is involved in the occurrence of arrhythmia. The method is based on the transgenic mouse, so the model can stably express the sponge to stably knock miRNA down and can inherit simultaneously. The mouse phenotype is stable, thus the mouse model is applicable for study of subsequent functions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for establishing a heart-specific microRNA knockdown mouse model. Background technique [0002] The vast majority of human diseases stem from abnormalities in gene expression. In recent years, a class of small molecule non-coding RNAs with post-transcriptional regulation of gene expression has been discovered, called "microRNAs (miRNAs)". Usually, miRNA negatively regulates the expression of a certain gene by binding to the 3′ untranslated region (UTR) of specific mRNA, leading to mRNA degradation or inhibition of translation process using mRNA as a template. The study of miRNA function is of great significance for further revealing the occurrence and development of diseases. [0003] When studying a specific miRNA, the strategy of "loss of function" is often adopted, such as "knockdown" (Knockdown, KD), that is, by a certain method to reduce the content of a specific miRN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63A01K67/027
Inventor 杨宝峰马宁吕延杰高旭潘振伟
Owner HARBIN MEDICAL UNIVERSITY
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