Regulating function of MR-1 gene in myocardial hypertrophy
A technique for cardiac hypertrophy and MR-1, which is used in gene therapy, cardiovascular system diseases, genetic material components, etc.
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Embodiment 1
[0064] : Establishment of MR-1 transgenic mouse model
[0065] Acquisition of MR1 gene: PCR reaction was carried out with primer A and primer B using a human skeletal muscle cDNA library (purchased from Clontech) as a template.
[0066] Primer A: 5'-ATTAGAATTCATGGCGGCGGTGGTAGCTGCT-3' (EcoRI)
[0067] Primer B: 5'-ATTAGTCGACTCAGGTCTGCACCCCAGACCC-3'(SalI)
[0068] The obtained PCR product contains MR1 gene, which is composed of 425 nucleotides and encodes 142 amino acids. This fragment was inserted into the vector pIRES2-GFP (Clontech. Company) with EcoRI and SalI restriction sites to construct the recombinant vector pIRES2-GFP-hMR-1. Then the recombinant vector was cut into a linear shape with SphI, and the 4.8 kb fragment was recovered by agarose gel electrophoresis, and purified by Nuclespin column (Clontech. Company). The linear DNA fragment containing MR1 gene was injected into 300 mouse fertilized eggs by microinjection technique, and 108 newborn mice were obtained. ...
Embodiment 2
[0070] : Transgenic mice perfused with angiotensin AngII
[0071] Take 14 MR1 transgenic mice and 13 non-transgenic mice as controls. Dissolve AngII in acetic acid to make (0.001-0.005mol / l) solution, subcutaneously inject 0.1-0.3mg AngII per kilogram of body weight per day, and perfuse it for 1-2 weeks. In addition, 14 transgenic mice and 13 non-transgenic mice were used as the sham operation group for control.
Embodiment 3
[0072] : Western blot hybridization detection of high expression of MR1 in transgenic mice
[0073] Extract transgenic mouse myocardial tissue protein, about 50 micrograms were electrophoresed in 10% polyacrylamide protein gel, transferred to nitrocellulose membrane, and MR1 antibody (self-made) and GAPDH antibody were used as internal references (Kangcheng Biological Company) for hybridization, and then used two The anti-peroxidase-conjugated antibody was used for color development and detected with a chemiluminescent kit (Amersham, Inc.). The technique of western blot hybridization showed that the MR1 gene was obviously highly expressed in the MR1 transgenic mice, while the MR1 gene was basically not expressed in the control mice (Figure 2).
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