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Construction method and application of mouse mammary tissue miRNA overexpression model

A breast tissue and construction method technology, applied in recombinant DNA technology, DNA/RNA fragments, other methods of inserting foreign genetic materials, etc. Infection risk, metabolism prevention, cost saving effect

Inactive Publication Date: 2020-11-24
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Combination of miRNA and mRNA leads to destabilization or degradation of transcripts, specifically causing decreased transcript levels

Method used

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  • Construction method and application of mouse mammary tissue miRNA overexpression model
  • Construction method and application of mouse mammary tissue miRNA overexpression model
  • Construction method and application of mouse mammary tissue miRNA overexpression model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of mouse mammary gland tissue miRNA overexpression model

[0047]The bta-16b-agomiR used in the present invention is based on the bta-miR-16b overexpression sequence with cholesterol and other modifications, and has the function of preventing miRNA from degrading in vivo; the ICR test mice are provided by the animal room of the Veterinary College of Yangzhou University.

[0048] The present embodiment provides a method for mouse mammary gland tissue miRNA overexpression model, comprising the following steps:

[0049] (1) Dilute bta-16b-agomiR to 330ng / uL with NaCl solution.

[0050] (2) Four ICR mice with similar body size and expected delivery date were randomly and equally divided into control group and bta-16b-agomiR solution treatment group. The experimental mice were raised in the aseptic animal room of Yangzhou University. They had free access to food and sufficient drinking water. The temperature was 20-28°C; the relative humidity was 45%-...

Embodiment 2

[0052] Example 2 Application of mouse mammary gland tissue miRNA overexpression model

[0053] (1) Inject 50uL NaCl solution into the mammary gland tissue of the mice in the control group through a micro-injector, and inject 50uL 330ng / uL of bta-16b-agomiR (prepared with NaCl solution) in the bta-16b-agomiR solution treatment group. The mice were put back into the mouse cage, and the newborn mice were not removed to maintain the normal lactation function of the mouse mammary glands.

[0054]

[0055] (2) 48 hours after the test treatment, the mice were killed by spinal dislocation, the skin was cut along the midline of the abdomen (without cutting the peritoneum), the breast area of ​​the test was exposed, the mammary gland tissue samples were taken out by blunt separation, and the damage of the mammary gland tissue of the mice was observed at the same time , including changes such as redness and swelling, leaving school, etc., and scored.

[0056] (3) Weigh 0.1 g of breas...

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Abstract

The invention belongs to the technical field of molecular biology, and relates to a construction method and application of a mouse mammary tissue miRNA overexpression model. A miRNA exogenous reagent-bta-16b-agomiR is injected through mammary tissue by adopting a microsyringe, a mouse is killed 48 hours after injection, RNA is extracted from the mammary tissue by using chloroform, reverse transcription is performed to obtain cDNA, and the overexpression effect of the bta-16b-agomiR and the mRNA expression levels of PGLYRP1 and PTX3 are detected by using fluorescent quantitative PCR; and the mammary tissue is subjected to HE staining and PGLYRP1 and PTX3 immunohistochemical tests. The method disclosed by the invention can be widely applied to overexpression of miRNA in the mouse mammary tissue and related research of the miRNA in living tissue, and a model basis is provided for tests of pathology mechanisms, immune mechanisms and the like of mastitis related diseases.

Description

technical field [0001] The present invention relates to the construction method and application of the miRNA overexpression model of mouse mammary gland tissue. A micro injector is used to inject miRNA exogenous reagents into the mouse mammary gland tissue to construct the miRNA overexpression model of mouse mammary gland tissue, which can be widely used The research on miRNA overexpression in mouse mammary gland tissue and miRNA in living tissue belongs to the field of molecular biotechnology. Background technique [0002] miRNA is a kind of highly conserved endogenous non-coding small RNA with a length of 19-25 nt. They are an important part of the biological gene regulatory network. Targets translation of mRNA, thereby regulating gene expression at the post-transcriptional level. Studies have found that miRNA plays an extremely important role in the immune regulation of innate immune response, pathogen infection, immunity and inflammation. [0003] The biological functi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89C12N15/113A01K67/027
CPCA01K67/0275A01K2217/05A01K2227/105A01K2267/0368C12N15/113C12N15/89C12N2310/141
Inventor 周静鹏杨章平陈志曹翔梁艳
Owner YANGZHOU UNIV
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