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Application of RSPO3 gene in sow ovarian granulosa cells

A granulosa cell, sow technology, applied in the field of cell engineering and genetic engineering, can solve the problem of unreported connection between growth and development

Active Publication Date: 2019-11-19
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of studies have shown that abnormal activation of the Wnt / β-catenin pathway can promote the proliferation, differentiation and migration of cancer cells, and most members of the Wnt family can be expressed in ovarian follicles, and the Frizzled protein receptor is involved in the early development of embryos and follicles , low-density lipoprotein-related receptors are mainly involved in ovulation and corpus luteum formation, but the relationship between RSPO3 gene and the growth and development of sow ovarian granulosa cells has not been reported yet

Method used

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  • Application of RSPO3 gene in sow ovarian granulosa cells
  • Application of RSPO3 gene in sow ovarian granulosa cells
  • Application of RSPO3 gene in sow ovarian granulosa cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 ChIP-Seq screening differential gene RSPO3

[0071] (1) Select follicles of two sizes with diameters of 3-5 and 7-10mm in porcine ovaries, formaldehyde crosslinks the entire tissue, connects the target protein with chromatin, separates genomic DNA, and breaks the DNA by ultrasonic waves; add target protein The specific antibody anti-H3K4me3 (SANTA CRUZ) forms an immunoprecipitation complex; decrosslinking and purifying DNA obtains a DNA sample of chromatin immunoprecipitation.

[0072] (2) DNA samples were sent to Shanghai Kangcheng Bioengineering Co., Ltd. for gene sequencing. The basic process of sequencing:

[0073] ①Sample quality assessment, using Quant-iT TM The dsDNA High-Sensitivity (HS) Assay Kit (Invitrogen) determined the purity and concentration of DNA samples.

[0074] ② Sequence library preparation, use TruSeq Nano DNA Sample Prep Kit (FC-121-4002, Illumina) to perform end repair, tail-end ligation and adapter ligation on DNA samples; use AMP...

Embodiment 2

[0080] Example 2 RNA extraction, quality detection and reverse transcription

[0081] (1) RNA extraction:

[0082] ① Sample digestion: extract RNA from tissue samples, take 50-100 mg of tissue samples, cut them up quickly on ice or repeatedly homogenize with a homogenizer, and add Trizol (50-100 mg / ml). Extract RNA from the cells without digesting the cells and add Trizol (10cm2 / mL) directly.

[0083] ② Place the tissue or cell sample on ice for 10-15 minutes, centrifuge at 12,000 g at 4°C for 5 minutes, and transfer the RNA-containing supernatant to a new RNase-free tube.

[0084] ③Add chloroform (the volume ratio of chloroform:Trizol is 1:5), shake vigorously, place at room temperature for 5 minutes, centrifuge at 12,000g at 4°C for 15 minutes, and carefully transfer the upper aqueous phase to a new RNase-free tube.

[0085] ④Add isopropanol (volume ratio of isopropanol:Trizol is 1:2), mix by inverting slightly, place at room temperature for 10min, centrifuge at 12,000g fo...

Embodiment 3

[0097] Example 3 qRT-PCR

[0098] 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:

[0099] 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﹚〉}

[0100] The detection gene uses GAPDH as an internal reference, and the qRT-PCR primers used in the present invention are:

[0101] qRT-PCR-RSPO3 Forward: 5′-CGTCAGTATTGTGCACTGTGA-3′;

[0102] Reverse: 5'-GGTGGGAGGACACAGGTTAC-3';

[0103] qRT-PCR-GAPDH Forward: 5′-GGACTCATGACCACGGTCCAT-3′;

[0104] Reverse: 5'-TCAGATCCACAACCGACACGT-3'.

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Abstract

The invention discloses an application of an RSPO3 gene in sow ovarian granulosa cells. Two kinds of follicles with diameters of 3-5 mm and 7-10 mm respectively are used as experimental materials, andthe relationship between H3K4me3 and the expression level of genes related to ovarian follicle development is studied by a sequencing technology. The enrichment degree of H3K4me3 in a promoter regionof the RSPO3 gene is found to be significantly different in the follicles with different size by ChIP-Seq; the expression quantity of the RSPO3 gene in the follicles with different size is found to be significantly different by qRT-PCR; promotion of the degree of the H3K4me3 is found to promote transcription of the RSPO3 gene by promoting or inhibiting the degree of H3K4me3 in the sow ovarian granulosa cells, and inhibition of the degree of H3K4me3 is found to inhibit transcription of the RSPO3 gene; the RSPO3 is found to promote the proliferation of the sow ovarian granulosa cells and inhibit the apoptosis of the cells through overexpression or interference of RSPO3.

Description

technical field [0001] The invention belongs to the technical fields of cell engineering and genetic engineering, and in particular relates to the application of RSPO3 gene in sow ovary granulosa cells. Background technique [0002] The ovary is the foundation of female breeding, its main function is to produce and discharge eggs, in addition, the granulosa cells in the ovarian follicles can secrete sex hormones, and promote the development and maintenance of female sexual characteristics. The length of the period of use. Epigenetics refers to changes in gene expression without changing the DNA double-stranded sequence, that is, changing the phenotype without changing the genotype. Among them, post-translational modification of histone occurs on each component of nucleosome octamer. In epigenetic research, H3K4me3 (3-methylation of lysine 4 on H3 histone, Tri-methylation of Histone H3 lysine 4) is a typical chromatin mark with transcriptional activity. Studies by Santos-Ro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N5/071C12N15/85
CPCC07K14/47C12N5/0682C12N15/85C12N2501/999C12N2800/107
Inventor 张哲袁晓龙何颖婷李加琪张豪
Owner SOUTH CHINA AGRI UNIV
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