The application of STAT3 in porcine ovarian granulosa cells

A technology of granulosa cells and ovaries, which is applied in the application field of STAT3 in porcine ovary granulosa cells, can solve the problem of high cost of live pig experiments, and achieve reliable results and well-designed effects

Active Publication Date: 2018-09-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the current research, the verification is only carried out at the level of cells and tissues, but not in live pigs. The main reason is that the change of a certain

Method used

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  • The application of STAT3 in porcine ovarian granulosa cells
  • The application of STAT3 in porcine ovarian granulosa cells
  • The application of STAT3 in porcine ovarian granulosa cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of STAT3 promoter reporter gene recombinant vector

[0038] (1) Use Primer5 to design primers, and use the extracted pig ear DNA as a template to amplify the 5' regulatory region of STAT3; the amplified fragments are purified and recovered, connected to the pMD18T vector (purchased from Takara), transformed, screened, sequenced and identified After being correct, the common plasmid was extracted and named as T-STAT3.

[0039] (2) BioEdit software analysis found that the 5' regulatory region sequence of STAT3 gene did not have MIu I and Xho I restriction endonuclease sites, but pGL3-basic Vector had MIu I and Xho I enzyme sites. Primer5 designed 5 pairs of primers for the missing fragment of the 5' regulatory region of the STAT3 gene, and the upstream and downstream primers were respectively added with MIu I and Xho I restriction site sequences. Using the T-STAT3 common plasmid as a template, PCR amplification; similarly, each missing fragment was...

Embodiment 2

[0053] Example 2 Culture and transfection of ovarian granulosa cells

[0054] (1) Collect healthy sow ovaries at Guangzhou Jiahe Slaughterhouse, place them in PBS buffer (on ice) containing 1% (v / v) double antibody, and quickly transport them back to the laboratory for processing;

[0055] (2) Wash the ovaries outside the cell room with PBS containing double antibodies, place them in a beaker, and put them into the transfer window;

[0056] (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;

[0057] (4) Centrifuge at 1000rpm for 5min, discard the supernatant, add 5mL PBS, mix by pipetting, and wash twice;

[0058] (5) Prepare complete medium: 89% DMEM, 10% serum and 1% double antibody, mix up and down;

[0059] (6) Add 5 mL of complete medium to resuspend the cell pellet;...

Embodiment 3

[0069] Embodiment 3qRT-PCR

[0070] 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:

[0071] 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﹚>}

[0072] The detection gene uses GAPDH as an internal reference, and the qRT-PCR primers used in the present invention are:

[0073] qRT-PCR-STAT3Forward: 5′-GGGCTTTATCAGTAAGGAGA-3′;

[0074] Reverse: 5'-GGAATGTCAGGGTAGAGGTA-3';

[0075] qRT-PCR-C / EBPβForward: 5′-CGGACTGCAAGCGGAAGGAGGA-3′;

[0076] Reverse: 5′-GGCTGGACGACGAGGATGTGGA-3′;

[0077] qRT-PCR-GAPDH Forward: 5′-TCCCGCCAACATCAAAT-3′;

[0078] Reverse: 5′-CACGCCCATCACAAAACAT-3′;

[0079] The total RNA of cells was extracted according...

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Abstract

The invention discloses an application of STAT3 in porcine ovarian granulosa cells. STAT3 is taken as a research object, and a porcine STAT3 gene promoter double-luciferase reporter gene recombinationplasmid is constructed, and the core promoter region of the STAT3 is found through the expression activity of the STAT3 gene promoter in the porcine ovarian granulosa cell; and then the interaction between the transcription factor C/EBP beta and the core promoter region of the STAT3 is verified; then C/EBP beta superexpression vector is constructed and small interfering RNA (C/EBP beta-siRNA) issynthesized, the effect of C/EBP beta on STAT3 is detected, finally, the expression vector of C/EBP beta and C/EBP beta-siRNA were transfected respectively to the granulosa cells in order to detect the apoptosis and proliferation of the cells. The application of STAT3 in porcine ovarian granulosa cells finds the application of the transcription factor C/EBP beta in the ovarian granulosa cell by finding the transcription factor C/EBP beta in the STAT3 promoter region, and has good application value for researching the ovarian follicular atresia mechanism.

Description

technical field [0001] The invention belongs to the technical fields of cell engineering and genetic engineering, and particularly relates to an application of STAT3 in porcine ovarian granulosa cells. Background technique [0002] The ovary is an important organ that determines the fertility of female animals, and one of its main functions is follicle development and ovulation. Abnormal ovarian function can lead to reduced reproduction, including premature ovarian failure (Premature ovarian failure, POF), polycystic ovarian syndrome (polycystic ovarian syndrome, PCOS) and ovarian cancer. Among them, the main causes of premature ovarian failure are: first, not enough primordial follicles are formed during the embryonic period (congenital deficiency); second, the activation and inhibition of primordial follicles are affected, and some studies have shown that the acceleration of follicular atresia can also cause premature ovarian failure. [0003] During the development of fo...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/113
CPCC07K14/4702C12N15/113C12N2310/14
Inventor 张哲袁晓龙李加琪周小枫
Owner SOUTH CHINA AGRI UNIV
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