Application of RSPO2 gene to porcine ovarian granulosa cells

A technology of granulosa cells and ovaries, applied in the application field of RSPO2 gene in porcine ovarian granulosa cells

Active Publication Date: 2021-09-14
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the relationship between RSPO2 gene and the growth and development of porcine ovarian granulosa cells has not been reported yet.

Method used

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  • Application of RSPO2 gene to porcine ovarian granulosa cells
  • Application of RSPO2 gene to porcine ovarian granulosa cells
  • Application of RSPO2 gene to porcine ovarian granulosa cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 constructs the overexpression vector of RSPO2

[0039] (1) Use Primer5 to design primers, and use the extracted cDNA of porcine granulosa cells as a template to amplify the CDS region of RSPO2; the amplified fragments are purified and recovered, connected to the pMD18T vector (purchased from Takara Company), transformed, screened, sequenced and identified After correct extraction of common plasmid.

[0040] (2) BioEdit software analysis found that there were no Hind III and Kpn I restriction endonuclease sites in the CDS region of RSPO2 gene, but there were Hind III and Kpn I restriction sites in the pcDNA3.1 vector. The upstream and downstream primers are respectively added with Hind III and Kpn I restriction site sequences. Using the recombinant pMD18T ordinary plasmid in the CDS region of RSPO2 as a template, PCR amplification; the fragment was purified and recovered, double-digested, connected to the pcDNA3.1 vector, transformed, screened, sequenced and ...

Embodiment 2

[0045] Example 2 Culture and transfection of ovarian granulosa cells

[0046] (1) Collect healthy sow ovaries, place them in PBS buffer solution (on ice) containing 1% double antibody, and quickly transport them back to the laboratory for processing;

[0047] (2) Wash the ovary 3-5 times outside the cell room with PBS containing 2% double antibody, place it in a beaker, and put it into the transfer window;

[0048] (3) Wipe the ultra-clean table of the cell room with alcohol, pick up the ovary with tweezers, draw the follicle fluid with a syringe, put it into a centrifuge tube containing 5mL of DMEM culture medium, and extract 9mL of the follicle fluid from each tube;

[0049] (4) Centrifuge at 1000rpm for 5min, discard the supernatant, add 5mL PBS, mix by pipetting, and wash twice;

[0050] (5) Prepare complete medium: 89% DMEM, 10% serum and 1% double antibody, mix up and down;

[0051] (6) Add 5 mL of complete medium to resuspend the cell pellet;

[0052] (7) First add 1...

Embodiment 3

[0061] Example 3 qRT-PCR

[0062] The qRT-PCR detection of genes in the present invention uses Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific Company). In the experiment, the comparative Ct value method was used to detect the content of the gene in the sample, and the specific calculation formula was as follows:

[0063] 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﹚>}

[0064] The detection gene uses GAPDH as an internal reference, and the qRT-PCR primers used in the present invention are:

[0065] qRT-PCR-RSPO2 Forward: 5′-GATGGAGACGCAGTAAGCGA-3′;

[0066] Reverse: 5′-CATATCTGGGGCTCGGTGTC-3′;

[0067] qRT-PCR-Caspase3 Forward: 5′-ACATGGAAGCAAATCAATGGAC-3′;

[0068] Reverse: 5'-TGCAGCATCCACATCTGTACC-3';

[0069] qRT-PCR-Caspase8 Forward: 5′-GAGCCTGGACTACATCCCAC-3′;

[0070] Reverse: 5'-GTCCTTCAATTCCGACCTGG-3';

[0071] qRT-PCR-Caspase9 Forward: 5′-GCTGAACCG...

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Abstract

The invention discloses application of an RSPO2 gene to porcine ovarian granulosa cells, and belongs to the technical field of cell engineering and gene engineering. According to the invention, RSPO2 is taken as a research object, an RSPO2 overexpression vector or RSPO2-siRNA are respectively transfected into ovarian granulosa cells by constructing the RSPO2 overexpression vector and synthesizing small interfering RNA (RSPO2-siRNA); then, the changes of signal channel gene mRNA and protein level related to ovarian granulosa cell apoptosis, proliferation and E2 secretion are detected; and finally, the phenotypic change of the ovarian granulosa cells is changed. The results show that RSPO2 can inhibit apoptosis of ovarian granulosa cells and promote proliferation and E2 secretion. By exploring the application of the RSPO2 to the ovarian granulosa cells, the invention has very good application value on researching an ovarian follicle atresia mechanism, initial estrus initiation and the like.

Description

technical field [0001] The invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to the application of RSPO2 gene in porcine ovary granulosa cells. Background technique [0002] Follicular development is a multicellular process involving oocyte maturation, granulosa cell proliferation, apoptosis, and hormone secretion. Increased granulosa cell death is likely a cellular mechanism that directly or indirectly impedes follicular development. For example, disruption of the estrogen receptor beta in mice results in reduced estrogen secretion from granulosa cells, hindered follicular maturation and ovulatory dysfunction. In the granulosa cells of mice with premature ovarian failure, up-regulating the expression of anti-Müllerian hormone can inhibit the apoptosis of granulosa cells and promote the development of follicles. [0003] RSPO2 (R-Spondin 2) is a key protein that activates the WNT signaling pathway, and the WNT s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12Q1/6888C12Q1/02G01N33/68
CPCC12N15/113C12Q1/6888G01N33/68G01N33/5005C12N2310/141C12Q2600/158
Inventor 张哲周小枫李加琪潘向春何颖婷袁晓龙张豪
Owner SOUTH CHINA AGRI UNIV
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