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Construction method for miRNA (micro Ribonucleic Acid) transgenic mouse model

A technology of transgenic mice and genes, applied in the field of transgenics, can solve the problems of unreported specific functions of the heart and increased expression levels

Active Publication Date: 2011-09-28
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Previous studies have shown that the expression level of miR-27b is significantly increased in mouse models of cardiac hypertrophy and human specimens of aortic sclerosis, suggesting that it may play an important role in the occurrence of cardiac diseases, but regarding its role in the heart The specific function has not yet been reported

Method used

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  • Construction method for miRNA (micro Ribonucleic Acid) transgenic mouse model
  • Construction method for miRNA (micro Ribonucleic Acid) transgenic mouse model
  • Construction method for miRNA (micro Ribonucleic Acid) transgenic mouse model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Construction of cardiomyocyte-specific miR-27b transgene vector

[0037] Digest the plasmid with α-MHC with Apa I and Sal I to obtain a 5.5kb fragment, remove the insulin gene promoter in the pRCH plasmid with Kpn I and Sal I, and remove the α-MHC promoter fragment Ligated with a promoterless pRCH fragment. The miR-27b genome sequence was amplified and ligated with primers added with Sal 1, Mlu1, Mlu 1, and EcoR 1, respectively, to obtain two copies of miR-27b fragments, which were ligated into the fragment digested with Sal 1 and EcoR 1. The above-mentioned vector obtained the transgenic vector of α-MHC-miR-27b-hGH containing three elements of the α-MHC promoter, 2 copies of the miR-27b gene and the human hGH gene ( figure 1 ).

Embodiment 2

[0038] Introduction of embodiment 2 transgenic vectors and screening of transgenic mice

[0039] After the transgenic vector was linearized by Kpn I and Sac II, a total of 210 fertilized eggs were injected and 200 eggs were transplanted by pronuclear microinjection of fertilized eggs. Three pseudopregnant female mice were pregnant and 13 offspring mice were obtained. . The results are shown in Table 1.

[0040] Table 1

[0041] Number of eggs injected

Number of eggs transferred

Number of mice transplanted

Number of pregnant mice

number of rats born

Number of positive mice

210

200

8

3

13

4

[0042] Genotype identification of the obtained transgenic mice:

[0043] (1) Preparation of mouse genomic DNA

[0044] 1. Cut the tail tip of 15-day-old mice about 0.5 cm into an Eppendorf tube.

[0045] 2. Add 400 μl of rat tail lysis buffer (0.5% SDS, 0.1M NaCl, 0.05 MEDTA, 0.01M Tris-Cl pH8.0, proteinase K, 2...

Embodiment 3

[0056] Example 3 Detection of miR-27b expression level in heart tissue of transgenic mice

[0057] (1) Northern blot detection of miR-27b expression

[0058] 1. Extraction of total tissue RNA

[0059] (1) Put 100 mg of mouse heart tissue into 2 ml Trizol, homogenize with a homogenizer, and incubate at room temperature for 5 minutes.

[0060] (2) Add 0.4ml of chloroform, close the cap tightly and shake vigorously for 15 seconds, then let it stand for 2-3 minutes. Centrifuge at 10000-12000g for 15 minutes at 2-8°C.

[0061] (3) Transfer the upper aqueous phase to a new centrifuge tube, add 0.5ml of isopropanol, mix well, leave at room temperature for 10 minutes, and centrifuge at 10000-12000g for 10 minutes at 2-8°C.

[0062] (4) Discard the supernatant, add at least 2ml of 75% isopropanol to wash, and centrifuge at no more than 7500g for 5 minutes at 2-8°C.

[0063] (5) Discard the supernatant, air dry for 5-10 minutes, add 500 μl of RNase-free water, and repeatedly suck to...

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Abstract

The invention provides a construction method for a miRNA (micro Ribonucleic Acid) transgenic mice model. In the construction method, a transgenic mouse with overexpression miR-27b in a myocardial cell is established by using a transgenic technology; the systematic phenotyping on the transgenic mouse finds that the mouse possibly has myocardial hypertrophy and heart function damage; the basic pathological changes of the mouse are similar to those of human hearts, therefore, with the establishment of the transgenic mouse model, a favorable animal model for researching the heart disease nosogenesis and researching and developing new drugs is provided.

Description

technical field [0001] The invention belongs to the technical field of transgenesis, and in particular relates to a method for constructing a miRNA transgenic mouse model. Background technique [0002] The heart is an important organ of the human body. As an important functional organ, the heart promotes blood flow, provides sufficient blood flow to organs and tissues, supplies oxygen and various nutrients, and takes away metabolic end products (such as carbon dioxide, urea and uric acid, etc.), so that cells maintain normal metabolism and function. In recent years, the incidence of heart disease has gradually increased. At present, 3 million people die of cardiovascular disease in my country every year, and it has become the number one killer of human health. Most people with heart disease eventually die from developing heart failure. The occurrence of heart disease is a complex process, and the molecular mechanism of the disease is still unclear, which may be the result ...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027A61K49/00
Inventor 杨晓王剑侯宁孙强张彦
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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