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Application of PIK3R2 in pig ovarian granular cells

A technology of granulosa cells and ovaries, which is applied in the application field of PIK3R2 in porcine ovary granulosa cells, can solve the problems of inability to verify live pigs and high cost, and achieve the effect of careful design and reliable results

Active Publication Date: 2016-06-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] This technology can only be verified at the cell level and tissue level, and cannot be verified at the individual level of live pigs. The main reason for this shortcoming is that the change of a phenotype of an animal is affected by many aspects. Whether a gene controls this The main gene of the phenotype still needs a lot of research to confirm, and the relationship between the target gene and miRNA is many-to-many, that is, one gene can also be the target of multiple miRNAs, and one miRNA can target multiple genes. It is found that there are very few major genes that can control the phenotype, that is, a phenotypic change in animals is difficult to be regulated by a gene or its target miRNA; The cost of in vivo experiments is too high

Method used

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  • Application of PIK3R2 in pig ovarian granular cells
  • Application of PIK3R2 in pig ovarian granular cells
  • Application of PIK3R2 in pig ovarian granular cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 The cultivation of ovarian granulosa cells

[0043] (1) Collect the ovaries at the slaughterhouse, put them in a thermos bottle at 37°C with PBS or normal saline (containing 1% double antibody), and quickly transport them back to the laboratory;

[0044] (2) After washing the collected ovaries with preheated PBS (containing 1% double antibody) for 3 times in a sterile culture room, they were quickly transferred to the ultra-clean workbench; a 1mL sterile disposable syringe was inserted shallowly into the cavity of the ovary Absorb follicular fluid from follicles;

[0045] (3) Place the aspirated follicular fluid in a 15 mL centrifuge tube containing an appropriate amount of DMEM, and centrifuge at room temperature for 6 min at 1000 rpm / min;

[0046] (4) Discard the supernatant, resuspend and centrifuge in DMEM, and wash the cells twice; prepare DMEM complete medium: 89% DMEM+10% FBS+1% double antibody;

[0047] (5) The cell resuspension and the complete m...

Embodiment 2

[0049] Example 2 Inoculation and transfection of ovarian granulosa cells

[0050] (1) The granulosa cells grow to about 90%, discard the medium, and wash 3 times with preheated PBS containing 1% double antibodies (the double antibodies are penicillin and streptomycin);

[0051] (2) Add trypsin for digestion, put it in the incubator for about 3 minutes, observe under the microscope until most of the cells float, immediately add the same amount of stop solution to stop the digestion;

[0052] (3) Wash 2 times with DMEM, centrifuge at 1000rpm / min for 5min;

[0053] (4) Gently resuspend the cell pellet with complete medium, evenly distribute into each well, supplement the volume with complete medium, shake gently, and culture in the incubator;

[0054] (5) About 24 hours, observe the state of granulosa cells, and prepare for transfection when the confluence of the cells reaches about 80%;

[0055] (6) transfection method is by Invitrogen company's 3000 kit instructions were ca...

Embodiment 3

[0058] Embodiment 3qRT-PCR

[0059] The qRT-PCR detection of gene and miRNA in the present invention adopts SYBRPremixExTaq kit and SYBRPrimeScript miRNART-PCRKit (TaKaRa company) respectively. In the experiment, the comparative Ct value method was used to detect the content of miRNA or gene in the sample, and the specific calculation formula was as follows:

[0060] Relative gene expression = 2-{-〈﹙Ct value of the target gene in the control group﹚-﹙Ct value of the internal reference gene in the control group﹚>}

[0061] The internal reference gene wherein detects miRNA with U6 as internal reference, and detects gene with GAPDH as internal reference. The qRT-PCR primers used in the present invention are:

[0062] qRT-PCR-BCL2Forward: 5'-GAAACCCCTAGTGCCATCAA-3';

[0063] Reverse: 5'-GGGACGTCAGGTCACTGAAT-3';

[0064] qRT-PCR-Caspase3Forward: 5′-GACTGTGGGATTGAGACG-3′;

[0065] Reverse: 5′-ACCCGAGTAAGAATGTGC-3′;

[0066] qRT-PCR-PIK3R2Forward: 5′-GGCAAGATCAACCGCACACAAG-3′;

...

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Abstract

The invention discloses application of PIK3R2 in pig ovarian granular cells. A PIK3R2 interference small fragment is synthesized with PIK3R2 as a research object, ovarian granular cells are transfected, it is found that PIK3R2 promotes granular cell apoptosis and inhibits cell proliferation, and research is performed by regulating the functions of miR-126-3p cells expressed by PIK3R2 in granular cells. After exogenous disturbance PIK3R2 is supplemented, cell function phenotype caused by miR-126-3p can be restored through the verification, it is shown that PIK3R2 is an important functional target of miR-126-3p in granular cells, and miR-126-3p can regulate cell development of the granular cells by means of PIK3R2. By means of PIK3R2 and application of targeted miRNA of miR-126-3p in ovarian granular cells, PIK3R2 has good application value for researching an ovarian follicles locking mechanism.

Description

technical field [0001] The invention belongs to the technical field of cell engineering and genetic engineering, and specifically relates to the application of PIK3R2 in porcine ovarian granulosa cells. Background technique [0002] In commercial pork production, the utilization period of sows is directly related to the economic benefits of pig farms. Factors that affect the utilization of sows mainly include reproductive barriers, natural environment, breed, nutrition, etc. Among them, reproductive barriers are one of the most important reasons for sows to be eliminated from the herd prematurely. Diseases of the ovaries are an important cause of reproductive failure in sows. [0003] The ovary is an important gonad in mammals and an important reproductive organ for follicular development and ovulation. As the basic unit of the ovary, the follicle maintains the existence, development and atresia of the egg. Granulosa cells are flat or cuboidal cells around the follicle, a...

Claims

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Application Information

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IPC IPC(8): C12N15/113
CPCC12N15/1137C12N2310/141
Inventor 李加琪张爱玲邓熙张哲张豪
Owner SOUTH CHINA AGRI UNIV
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