Application of RBP1 gene in sow ovarian granular cells

A technology of granulosa cells and sows, which is applied in the field of cell engineering and genetic engineering, and can solve the problems that the relationship between RBP1 gene and the growth and development of sow ovary granulosa cells has not been reported.

Active Publication Date: 2019-10-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In cancer patients, RBPs participate in AKT/EGFR, Wnt and other signaling pathways, and affect the occurrence and development of cancer by regulating cell prolif

Method used

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  • Application of RBP1 gene in sow ovarian granular cells
  • Application of RBP1 gene in sow ovarian granular cells
  • Application of RBP1 gene in sow ovarian granular cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 ChIP-Seq screening of differential gene RBP1

[0069] (1) Select two follicles with a diameter of 3-5 and 7-10mm in the sow ovary, formaldehyde cross-links the entire tissue, that is, connects the target protein with chromatin, separates the genomic DNA, and breaks the DNA by ultrasound; add the target protein The specific antibody anti-H3K4me3 (SANTA CRUZ) forms an immunoprecipitation complex; decrosslinking and purifying the DNA obtains a DNA sample of chromatin immunoprecipitation.

[0070] (2) DNA samples were sent to Shanghai Kangcheng Bioengineering Co., Ltd. for gene sequencing. The basic process of sequencing:

[0071] ①Sample quality assessment, using Quant-iT TM The dsDNA High-Sensitivity (HS) Assay Kit (Invitrogen) determined the purity and concentration of DNA samples.

[0072] 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; ...

Embodiment 2

[0078] Example 2 RNA extraction, quality detection and reverse transcription

[0079] (1) RNA extraction:

[0080] 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 cells without digesting cells, directly add Trizol (10cm 2 / mL).

[0081] 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.

[0082] 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.

[0083] ④Add isopropanol (volume ratio of isopropanol:Trizol is 1:2), mix by inverting slightly, place at room temperature for 10min, centrifuge at 12,000g for 15min ...

Embodiment 3

[0095] Example 3 qRT-PCR

[0096] The qRT-PCR detection of the genes in the present invention uses the Maxima SYBR Green qPCR Master Mix (2X) kit (Thermo Scientific). 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:

[0097] 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﹚>}

[0098] The detection gene uses GAPDH as an internal reference, and the qRT-PCR primers used in the present invention are:

[0099] qRT-PCR-RBP1 Forward: 5′-TAGGGTTCATTGTGGTCAGTGT-3′;

[0100] Reverse: 5'-CAATTTTGCGCAAGGCCACA-3';

[0101] qRT-PCR-GAPDH Forward: 5′-GGACTCATGACCACGGTCCAT-3′;

[0102] Reverse: 5'-TCAGATCCACAACCGACACGT-3'.

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Abstract

The invention discloses application of a RBP1 gene in sow ovarian granular cells. RBP1 is taken as a research object, and a molecular and cell biological method is adopted to study the application ofthe RBP1 in sow ovarian granular cells. The molecular and cell biological method includes the following steps that through ChIP-Seq, it is found that the enrichment degree of H3K4me3 is different in RBP1 gene promoter regions in follicles different in size; through qRT-PCR, it is found that the expression quantity of the RBP1 gene in follicles different in size is different significantly. By promoting or inhibiting the enrichment degree of H3K4me3 in the sow ovarian granular cells, it is found that promotion of the enrichment degree of H3K4me3 can promote transcription of the RBP1 gene, and inhibition of the enrichment degree of H3K4me3 can inhibit transcription of the RBP1 gene; through RBP1 over-expression or interference with RBP1, it is found that RBP1 can promote proliferation of thesow ovarian granular cells and inhibit 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 RBP1 gene in sow ovary granulosa cells. Background technique [0002] The follicle is the basic structural and functional unit of the ovary, and its main function is to ovulate and secrete hormones. Excessive apoptosis of granulosa cells in follicles can induce follicular atresia, reduce the frequency of estrus in female animals, and thus affect female productivity. Histones are the chromatin structural proteins of eukaryotes. Basic histones can kink with the negatively charged DNA double helix structure to form nucleosomes. Histone post-translational modification is a similar regulation of chromatin "opening" The unique mode of "turning off" or "off", reprograms the genome of living organisms by changing the chromatin structure, regulating the transcription and expression of genes in the nucleus, for example, hist...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/85C12N15/113C12Q1/6888
CPCC07K14/47C12N15/85C12N15/113C12Q1/6888C12N2800/107C12N2310/14C12Q2600/158
Inventor 李加琪何颖婷张哲袁晓龙张豪
Owner SOUTH CHINA AGRI UNIV
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