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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

Pending Publication Date: 2021-10-22
NORTHEAST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the stock of beef cattle in my country is high, the carcass weight of beef cattle is far below the world average level, and the demand is greater than the supply, and the price keeps rising, which affects people's consumption demand
Among them, muscle development is the key factor affecting the quality and yield of beef cattle. The current research has not fully revealed the mechanism of myogenic differentiation, and since the establishment of bovine myoblast cell lines, there has been a lack of effective differentiation methods, and the differentiation efficiency is low and uneven. , therefore, it is crucial to discover efficient methods for bovine myoblast differentiation

Method used

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  • Method for promoting bovine myoblast differentiation by using miR-18 inhibitor
  • Method for promoting bovine myoblast differentiation by using miR-18 inhibitor
  • Method for promoting bovine myoblast differentiation by using miR-18 inhibitor

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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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Abstract

The invention discloses a method for promoting bovine myoblasts to differentiate by using miR-18 inhibitor, relates to a method for promoting bovine myoblasts to differentiate by using an miR-18 inhibitor, and discloses a novel and efficient method for promoting bovine myoblasts to differentiate by using the miR-18 inhibitor, which comprises bovine myoblasts recovery culture, subculture and differentiation culture technologies. According to the method, the miR-18 inhibitor is transfected into the bovine myoblasts, and immunostaining identification, fluorescent quantitative PCR identification and the like are utilized after culture, so that the miR-18 inhibitor is found to have an obvious bovine myoblast differentiation promoting effect, and a high-efficiency differentiation result is obtained. The method is applied to the field of bovine myoblast differentiation promotion.

Description

technical field [0001] The invention relates to a method for promoting differentiation of bovine myoblasts by using miR-18 inhibitors. Background technique [0002] Skeletal muscle is the most widely distributed important tissue and organ in mammals, which can maintain the normal movement and energy metabolism balance of the body. Exploring the mechanism of muscle cell proliferation, differentiation and regeneration is crucial to improving animal husbandry performance and revealing various muscle diseases in animals and humans. In animal husbandry production, the beef cattle industry is an important part. With the development of the national economy, people's living conditions have been greatly improved, and the demand for beef has gradually increased. Since the 1990s, my country's beef output has become the third largest country after the United States and Brazil. Although the stock of beef cattle in my country is high, the carcass weight of beef cattle is far lower than ...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0658C12N2501/999
Inventor 滕春波梁洋孟博文孔德麟
Owner NORTHEAST FORESTRY UNIVERSITY