Application of rspo3 gene in sow ovary granulosa cells

A technology of granulosa cells and sows, applied in the field of cell engineering and genetic engineering, can solve the problems of unreported relationship between growth and development, and achieve the effects of careful design, promotion of proliferation and inhibition of apoptosis

Active Publication Date: 2022-05-10
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 ovary granulosa cells
  • Application of rspo3 gene in sow ovary granulosa cells
  • Application of rspo3 gene in sow ovary 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] ③ Add 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 f...

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 the application of RSPO3 gene in sow ovary granulosa cells. The invention uses two kinds of follicles with a diameter of 3-5 mm and 7-10 mm as experimental materials, and uses sequencing technology to study the relationship between H3K4me3 and expression levels of genes related to ovarian follicle development. Through ChIP-Seq, it was found that the enrichment degree of H3K4me3 in the promoter region of RSPO3 gene was significantly different in follicles of different sizes; through qRT-PCR, it was found that the expression of RSPO3 gene in different sizes of follicles was significantly different; by promoting or inhibiting maternal The level of H3K4me3 in porcine ovarian granulosa cells was found to promote the level of H3K4me3 can promote the transcription of RSPO3 gene, and inhibit the level of H3K4me3 can inhibit the transcription of RSPO3 gene; by overexpressing or interfering with RSPO3, it was found that RSPO3 can promote the proliferation of sow ovary granulosa cells and inhibit cell apoptosis .

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 Patents(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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