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
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Examples
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
Description
Claims
Application Information
- IPC
- C07K14/47; C12N5/071; C12N15/85
- CPC
- C07K14/47; C12N5/0682; C12N15/85; C12N2501/999; C12N2800/107
- Inventors
- 张哲; 袁晓龙