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Application of DNMT1 gene in porcine ovarian granulosa cells

A technology of granulosa cells and ovaries, applied in the application field of DNMT1 gene in porcine ovary granulosa cells, to achieve reliable results and well-designed effects

Pending Publication Date: 2020-10-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of DNA methylation or DNMT1 on porcine ovarian granulosa cells has not been reported yet.

Method used

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  • Application of DNMT1 gene in porcine ovarian granulosa cells
  • Application of DNMT1 gene in porcine ovarian granulosa cells
  • Application of DNMT1 gene in porcine ovarian granulosa cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Culture of porcine ovary granulosa cells pGCs

[0041] (1) In vitro isolation and primary culture of pGCs

[0042] ① Collect the ovaries of 100-120kg sows from the slaughterhouse, select the ovaries with bright appearance, normal development, no corpus luteum, many antral follicles, and fullness, and store them in ovaries containing 1% (w / v) double antibody (penicillin and streptomycin). mixed solution, the same below) in 1×PBS reagent, placed on ice and quickly brought back to the laboratory.

[0043] ②Transfer the ovary samples to the sterile laboratory, wash the ovaries several times with 1×PBS containing 1% double antibody, and wipe the ultra-clean bench and key experimental equipment sterilized by ultraviolet radiation in advance with 75% (v / v) ethanol.

[0044] ③Use tweezers to pick up the ovary, and use a 1mL disposable sterile syringe to draw 2mL of follicular fluid from 3-5mm follicles into a 15mL sterile centrifuge tube containing 5mL of high-glucos...

Embodiment 2

[0056] Example 2 drug treatment of pGCs

[0057] The drug treatment pGCs of the present invention is implemented as follows:

[0058] (1) Observe the state of the cells. When the confluence of the cells reaches about 80%, discard the medium, and wash the culture flask 2 to 3 times with preheated 1×PBS (containing 1% double antibody).

[0059] (2) Add an appropriate amount of 0.25% trypsin to the culture flask to digest the cells in the cell culture flask for 3-5 minutes, stop the digestion when most of the cells fall off, and use preheated 1×PBS (containing 1% double antibody) Wash the cells 2 times.

[0060] (3) Resuspend the cells with complete medium (high-glucose DMEM containing 10% (v / v) calf serum and 1% double antibody), add the cell suspension evenly into the cell culture wells, and culture the wells according to different sizes Supplement the complete medium with the appropriate amount, and shake gently evenly.

[0061] (4) Placed at 37°C, 5% CO 2 After 24 hours, ...

Embodiment 3

[0065] Example 3 pGCs total RNA extraction

[0066] The present invention uses Trizol as a lysate to extract cellular RNA (operate with reference to the instructions of the Trizol kit of Takara Company), and the steps are as follows:

[0067] (1) First wash the wells of the cell culture plate in Example 2 with PBS for 1-2 times, then add 1 mL Trizol to each well, place on ice (or refrigerator at 4°C) to fully lyse and digest the cells for 5 min, and pipette with a pipette tip.

[0068] (2) Transfer the lysed cell suspension to a 1.5mL centrifuge tube, add 200μL chloroform, shake vigorously for 15s, incubate at room temperature for 3-5min, and centrifuge at 12,000×g for 15min at 4°C.

[0069] (3) Carefully pipette the upper layer of colorless liquid into a new 1.5mL centrifuge tube, add an equal volume of isopropanol to the supernatant and mix well (about 500μL), let stand at 4°C for 15-20min, then place at 4°C Centrifuge at 12,000×g for 10-15 minutes under the same conditions...

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Abstract

The invention discloses an application of a DNMT1 gene in porcine ovarian granulosa cells, belonging to the technical fields of cell engineering and gene engineering. According to the invention, the influence of DNMT1 on pGCs proliferation, apoptosis and E2 secretion is explored from a cellular level and a molecular level by taking a catalytic enzyme DNMT1 formed by DNA methylation as an entry point. Results show that the DNMT1 gene participates in promotion of proliferation of granulosa cells and E2 secretion and inhibition of apoptosis and maturation of the granulosa cells. The invention provides an important value for researching the influence mechanism of DNA methylation on ovarian follicle development and sow reproductive performance.

Description

technical field [0001] The invention belongs to the technical fields of cell engineering and genetic engineering, and in particular relates to the application of DNMT1 gene in porcine ovarian granulosa cells. Background technique [0002] As we all know, the production and reproduction efficiency of sows has a great impact on the economic benefits of modern intensive pig breeding production. About 10% to 30% of selected breeding gilts are eliminated due to lack of heat or easy pregnancy failure. In recent years, domestic and foreign scholars Much work has been carried out in both basic research and practical applications in the production of reproductive traits. 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 initiation of primordial follicle growth. Apoptosis of porcine granulosa cells (pGCs) is the mai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61P15/00A61P15/08C12Q1/6851C12Q1/6888G01N33/68
CPCA61K48/005A61P15/00A61P15/08C12Q1/6888C12Q1/6851G01N33/68C12Q2600/124C12Q2600/158C12Q2600/154C12Q2531/113C12Q2561/113
Inventor 袁晓龙何波李加琪张哲张豪
Owner SOUTH CHINA AGRI UNIV
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