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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

AI Technical Summary

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, it is basically difficult to verify at the individual level of live pigs; In addition, in the absence of experiments with a high degree of confidence in success, the cost of using pigs for live experiments is too high

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
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 The cultivation of ovarian granulosa cells

[0040] (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;

[0041] (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;

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

Embodiment 2

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

[0047] (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);

[0048] (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;

[0049] (3) DMEM was washed twice, and centrifuged at 1000rpm for 5min during this period;

[0050] (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;

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

[0052] (6) transfection method is by Invitrogen company's 3000 kit...

Embodiment 3

[0056] Embodiment 3qRT-PCR

[0057] The qRT-PCR detection of gene and miRNA in the present invention adopts SYBRPremixExTaq kit and SYBRPrimeScriptmiRNART-PCRKit of 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:

[0058] Relative gene expression = 2 -{〈﹙实验组目的基因Ct值﹚-﹙实验组内参基因Ct值﹚〉-〈﹙对照组目的基因Ct值﹚-﹙对照组内参基因Ct值﹚〉}

[0059] For the internal reference gene, U6 is used as an internal reference for miRNA detection, and GAPDH is used as an 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′-CACCACCACAGAGCAGGC...

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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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