Method for promoting bovine myoblast differentiation by using miR-18 inhibitor
A technology of mir-18 and myoblasts, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of low differentiation efficiency, impact on consumer demand, large demand, etc., and achieve the effect of improving differentiation efficiency
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specific Embodiment approach 1
[0025] Specific embodiment one: In this embodiment, a method for promoting differentiation of bovine myoblasts using miR-18 inhibitors is implemented in the following steps:
[0026] 1. Subculture of bovine myoblasts
[0027] Bovine myoblasts were recovered and cultured until 70%-80% of the cells were confluent, and subcultured;
[0028] 2. Preparation of transfection reagents
[0029] Take centrifuge tubes A and B, add 50uL Opti-MEM serum-free medium and 1.5uL miR-18 inhibitor premix to tube A; add 50uL Opti-MEM serum-free medium and 1uL RNAiMAX to tube B, let stand for 5 minutes, put A Mix the reagents in the tube and add to B, pipette 3 times, and let it stand at room temperature for 20 minutes to obtain the transfection reagent;
[0030] 3. Transfection
[0031] Bovine myoblasts were subcultured to the third generation, plated and subcultured, added proliferation medium and cultured for 24 hours, then replaced with differentiation medium, then added transfection reagent...
specific Embodiment approach 2
[0033] Specific embodiment two: the difference between this embodiment and specific embodiment one is that the bovine myoblast recovery method is as follows: take frozen bovine myoblasts, shake and melt them in a water bath at 37 degrees, and remove the supernatant after centrifugation at 1000 rpm for 5 minutes , resuspended cells with 1mL proliferation medium, inoculated in 6cm cell culture dish, added proliferation medium to 3mL, and cultured in 37°C incubator. Other steps and parameters are the same as those in Embodiment 1.
specific Embodiment approach 3
[0034] Specific embodiment three: the difference between this embodiment and specific embodiment one or two is that the proliferation medium formula: DMEM high glucose cell culture medium (DMEM-HIGH GLUCOSE medium)+15% fetal bovine serum (ES-FBS)+ 10% horse serum (HSR) + 1% double antibody + 1% glutamine substitute. Other steps and parameters are the same as those in Embodiment 1 or Embodiment 2.
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