Regulation effect of miR-450b-3p on polyester of adipocytes and application of miR-450b-3p
A technology of mir-450b-3p and 1.mir-450b-3p is applied in the application field of polyester inhibition of fat cells, and can solve the problems of affecting economic benefits, low intramuscular fat content, affecting meat quality and the like
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Embodiment 1
[0070] Example 1, found that miR-450b-3p may be related to adipocyte polyester
[0071] 1. Expression characteristics of miRNA in adipocytes
[0072] 1. Preparation of porcine preadipocytes
[0073] Washing solution (pH7.2-7.4): NaCl 2g, KCl 0.05g, NaCl 2 HPO 4 12H 2 O 0.725g, KH 2 PO 4 0.05g, dilute to 250mL with water, add penicillin and streptomycin after autoclaving, so that the concentration of penicillin is 100IU / mL, and the concentration of streptomycin is 100μg / mL, and set aside at 4°C.
[0074] Digestive solution: 0.1g type I collagenase, 2g bovine serum albumin, dissolved in 100mL ultrapure water, filtered through a 0.22μm filter membrane, collected filtrate, 10mL / tube for aliquot, and stored at -20°C for later use.
[0075] The longissimus dorsi muscle of 3-day-old male piglets of Jiaxing black pigs was collected under aseptic conditions, washed with washing solution, and then cut into about 1mm 3 Tissue block; take the tissue block, immerse in the digestive...
Embodiment 2
[0088] Example 2. Overexpression of miR-450b-3p promotes adipocyte polyester
[0089] The tested mimics are: miR-450b-3p-mimics, NC-mimics.
[0090] 1. Inoculate 3T3-L1 cells in a 12-well cell culture plate, adopt DMEM high-glucose medium containing 1% (volume ratio) penicillin / streptomycin solution and 10% (volume ratio) FBS (replace once every 1d) Medium), placed at 37°C, 5% CO 2 Culture in an incubator until the cells reach 80-90% confluency.
[0091] 2. After completing step 1, discard the supernatant and replace with fresh DMEM / F12 complete medium, 1ml / well.
[0092] DMEM / F12 complete medium: DMEM / F12 medium containing 1% (volume ratio) penicillin / streptomycin solution and 10% (volume ratio) FBS.
[0093] 3. Take 60μl 1×riboFECT TMCP Buffer and 5μl 20μM mimics solution to be tested, mix gently, then add 6μl riboFECT TM CP Reagent, gently pipette to mix, and incubate at room temperature for 15 minutes to obtain the transfection complex.
[0094] 4. After completing s...
Embodiment 3
[0105] Example 3. Knockdown of miR-450b-3p inhibits adipocyte polyester
[0106] The tested mimics are: miR-450b-3p-inhibitor, NC-inhibitor.
[0107] 1. Inoculate 3T3-L1 cells in a 12-well cell culture plate, adopt DMEM high-glucose medium containing 1% (volume ratio) penicillin / streptomycin solution and 10% (volume ratio) FBS (replace once every 1d) Medium), placed at 37°C, 5% CO 2 Culture in an incubator until the cells reach 80-90% confluency.
[0108] 2. After completing step 1, replace with fresh DMEM / F12 complete medium, 1ml / well.
[0109] 3. Take 60μl 1×riboFECT TM CP Buffer and 5μl 20μM mimics solution to be tested, mix gently, then add 6μl riboFECT TM CP Reagent, gently pipette to mix, and incubate at room temperature for 15 minutes to obtain the transfection complex.
[0110] 4. After completing step 2, add 71 μl of the transfection complex prepared in step 3 to each well and incubate for 48 hours.
[0111] 5. After step 4 is completed, the cells are taken to i...
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