Application of tri-methylation of histone H3lysine 4( H3K4me3) to granulosa cells of pigs

A technology of histone methylation and granulosa cells, applied in the field of cell engineering and genetic engineering, to achieve the effect of good application value, detailed design and reliable results

Active Publication Date: 2019-06-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the effect of histone methylation or demethylation modi...

Method used

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  • Application of tri-methylation of histone H3lysine 4( H3K4me3) to granulosa cells of pigs
  • Application of tri-methylation of histone H3lysine 4( H3K4me3) to granulosa cells of pigs
  • Application of tri-methylation of histone H3lysine 4( H3K4me3) to granulosa cells of pigs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 The cultivation of porcine ovary granulosa cells

[0046] (1) The pig ovary granulosa cells of the present invention are collected from Jiahe Wanggang Slaughterhouse, and the ovaries of slaughtered sows are collected and stored in PBS reagent containing 1% double antibodies (penicillin and streptomycin), and brought back to the laboratory at low temperature for processing;

[0047] (2) Wash the ovary several times with PBS containing 1% double antibody, and transfer the sample to the cell room;

[0048] (3) UV-irradiate the ultra-clean bench of the cell room for 20 minutes in advance, and wipe it with 75% alcohol;

[0049] (4) Pick up the ovary with tweezers, use a 1mL syringe to draw follicle fluid into 5mL DMEM medium, and absorb follicle fluid to 9mL in each tube;

[0050] (5) Centrifuge at 800rpm for 5min, discard the supernatant, add preheated PBS to gently pipet and pellet the cells, and wash twice.

[0051] (6) For culture in a 75mL cell culture fl...

Embodiment 2

[0054] Example 2 explores the concentration of drugs used for H3K4me3 agonists and inhibitors

[0055] (1) Digest the cultured cells with trypsin, put them in 37°C, 5% CO 2 Incubator for 3-5 minutes, when most of the cells were observed to be suspended under the microscope, immediately add an equal amount of complete medium to stop the digestion;

[0056] (2) Collect the cell suspension and centrifuge at 800rpm for 5min, wash the cells twice with preheated PBS, resuspend with complete medium, add the cell suspension evenly into a 6-well plate, shake gently evenly, put in 37°C, 5 %CO 2 Cultivate in the incubator for 24h.

[0057] (3) Observe the state of the cells. When the confluence of the cells reaches about 80%, the cells are treated with H3K4me3 inhibitor (BCl-121) and H3K4me3 agonist (PBIT) drugs, and subsequent experiments are carried out.

[0058] (4) The cells were treated with H3K4me3 inhibitor (BCl-121) and H3K4me3 agonist (PBIT) at concentrations of 50 μM, 75 μM ...

Embodiment 3

[0061] Embodiment 3Western Blot

[0062] (1) Extraction of total cell protein (using P1250 total protein extraction kit from Beijing Pulilai Gene Technology Co., Ltd.):

[0063] ①Digest and wash the cells, collect the cells by centrifugation at 800rpm, every 5-10*10 6 Add 0.5mL lysate to the cells, shake and resuspend, 4°C for 2 minutes;

[0064] ②Add 1mL of extraction reagent to every 0.5mL of lysate in proportion, shake and mix well, and let stand at 4°C for 10min;

[0065] ③ Centrifuge at 10,000rpm at 4°C for 10min. The solution is divided into upper and lower phases. Absorb the upper phase and absorb the lower phase as much as possible to collect the protein flocs in the middle of the two-phase solution. If the phases cannot be separated, mix again with 50μL distilled water and centrifuge ;

[0066] ④ Add 1 mL of ethanol to wash the precipitate, centrifuge at 10,000 rpm at 4°C for 3 min, remove all liquid in the tube, and dry at room temperature.

[0067] (2) SDS-PAGE:...

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Abstract

The invention discloses an application of tri-methylation of histone H3lysine 4( H3K4me3) to granulosa cells of pigs, and belongs to the technical field of cell engineering and genetic engineering. H3K4me3 is used as a penetration point, and a cell biology method is used for researching the influence of the H3K4me3 to the functions of the granulosa cells of pigs. The technical scheme is detailed in design, and reliable in results. In order to confirm the influence of H3K4me3 to the functions of the granulosa cells of the pigs, verification is performed from multiple levels and multiple angles,and verification is performed from the cell level and the protein level. The invention explains that the H3K4me3 has influence on the functions of the granulosa cells of the pigs, the H3K4me3 can promote proliferation of the granulosa cells of the pigs, can restrain the apoptosis of the granulosa cells of the pigs, and can reduce the proportion of the granulosa cells of the pigs, blocked in the GO/G1 period. The application has a good application value of research of histone methylation on an influence mechanism on the development of granulosa cells and the reproductive performance of sows.

Description

technical field [0001] The invention belongs to the technical field of cell engineering and genetic engineering, and in particular relates to the application of histone methylation H3K4me3 in porcine ovarian granulosa cells. Background technique [0002] The production and reproduction efficiency has a great influence on the economic benefits of pork. About 10% to 30% of the selected breeding gilts are eliminated due to lack of heat or easy pregnancy failure. A lot of work has been carried out. As far as female animals are concerned, the ovary is the basis for the normal reproduction of sows. The growth and differentiation of follicles is an important guarantee for ovarian function. The differentiation and growth of granulosa cells are the key to the growth of primordial follicles. The apoptosis of porcine ovarian granulosa cells is the main cause of follicular atresia. When the follicle atresia occurs, the estrogen produced by the granulosa cells decreases, and the progest...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
Inventor 袁晓龙何颖婷张豪张哲钟玉宜
Owner SOUTH CHINA AGRI UNIV
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