Application of miR-126-3p to porcine ovarian granular cells

A technology of granulosa cells and ovaries, which is applied in the application field of miR-126-3p in porcine ovary granulosa cells, can solve the problems of high cost and difficult verification at the individual level of living pigs, and achieve reliable results

Active Publication Date: 2016-05-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In the current research, since the relationship between miRNA and target gene is many-to-many, and a phenotypic change in animals is difficult to control through the regulation of a miRNA and its target gene, i

Method used

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  • Application of miR-126-3p to porcine ovarian granular cells
  • Application of miR-126-3p to porcine ovarian granular cells
  • Application of miR-126-3p to porcine ovarian granular cells

Examples

Experimental program
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Example Embodiment

[0039] Example 1 Cultivation of ovarian granulosa cells

[0040] (1) Collect the ovaries in the slaughterhouse, place them in a thermos flask at 37°C with PBS or normal saline (containing 1% bi-antibody) and quickly transport them back to the laboratory;

[0041] (2) Wash the collected ovaries in a sterile culture room with preheated PBS (containing 1% bi-antibody) for 3 times, then quickly transfer to the ultra-clean workbench; use a 1mL sterile disposable syringe to shallowly insert the ovarian cavity Absorb follicular fluid from the follicle;

[0042] (3) Put the aspirated follicular fluid in a 15mL centrifuge tube containing an appropriate amount of DMEM, and centrifuge at 1000rpm for 6min at room temperature;

[0043] (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;

[0044] (5) Aspirate the cell resuspension and complete medium to inoculate in a 75mL culture flask; place...

Example Embodiment

[0046] Example 2 Seeding and transfection of ovarian granulosa cells

[0047] (1) Granule cells grow to about 90%, discard the medium, and wash 3 times with pre-warmed PBS containing 1% double antibody (the double antibody is penicillin and streptomycin);

[0048] (2) Add trypsin for digestion, put it in the incubator for about 3 minutes, observe under the microscope that most of the cells float up, immediately add the same amount of stop solution to stop the digestion;

[0049] (3) DMEM was washed twice, during which time it was centrifuged at 1000 rpm for 5 minutes;

[0050] (4) Gently resuspend the cell pellet in complete medium, evenly divide it into each well, replenish the volume with complete medium, shake gently, and place it in an incubator for culture;

[0051] (5) About 24 hours, observe the state of granular cells, and prepare for transfection when the cell confluence reaches about 80%;

[0052] (6) The transfection method is according to Invitrogen's 3000 kit instructions i...

Example Embodiment

[0056] Example 3 qRT-PCR

[0057] The qRT-PCR detection of genes and miRNA in the present invention adopts SYBRPremixExTaq kit and SYBRPrimeScriptmiRNART-PCRKit of TaKaRa Company, respectively. The experiment adopts the comparative Ct value method to detect the content of sample miRNA or gene, and the specific calculation formula is as follows:

[0058] Relative gene expression = 2 -{-}

[0059] For the internal reference genes, U6 is used as internal reference for miRNA detection, and GAPDH is used as internal reference for gene detection. The qRT-PCR primers used in the present invention are:

[0060] qRT-PCR-BCL2Forward: 5′-GAAACCCCTAGTGCCATCAA-3′;

[0061] Reverse: 5'-GGGACGTCAGGTCACTGAAT-3';

[0062] qRT-PCR-Caspase3Forward: 5′-GACTGTGGGATTGAGACG-3′;

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

[0064] qRT-PCR-PIK3R2Forward: 5'-GGCAAGATCAACCGCACACAAG-3';

[0065] Reverse: 5'-CACCACCACAGAGCAGGCAT-3';

[0066] qRT-PCR-TSC1Forward: 5′-GACCCATATCTATGCGGACCC-3′;

[0067] Reverse: 5'-TGCTGG...

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Abstract

The invention discloses application of miR-126-3p to porcine ovarian granular cells and belongs to the technical field of cell engineering and gene engineering. According to the application, miR-126-3p serves as a research object, and a cell biological method is adopted for studying the application of miR-126-3p to the porcine ovarian granular cells. The application of miR-126-3p and PIK3R2 to the porcine ovarian granular cells and the targeted relation between miR-126-3p and the genes PIK3R2 and TSC1 in the porcine ovarian granular cells are proved for the first time; cell function phenotypes caused by miR-126-3p can be replied by supplementing exogenous interferential PIK3R2 and TSC1, and it is further indicated that PIK3R2 and TSC1 are an important functional target of miR-126-3p in the granular cells, and miR-126-3p can regulate development of the granular cells through PIK3R2 and TSC1.

Description

technical field [0001] The invention belongs to the technical field of cell engineering and genetic engineering, and specifically relates to the application of miR-126-3p 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, breeds, nutrition, diseases, 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 ...

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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