Application of transcription factor CEBP alpha serving as transcription factor of Kiss1 promoter region

A technology of transcription factor and promoter region, applied in the field of genetic engineering and cell engineering, can solve the problems of small testis, reduced testis and abnormal follicle development in male mice, and achieve the effect of complete results and rich experimental content.

Active Publication Date: 2018-10-30
SOUTH CHINA AGRI UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

The study found that the allele mutation of Kiss1 significantly reduced the number of follicles in rats and slowed down the development of ovarian follicles. In the late stage of ovarian follicle development, with the increase of estrogen, Kiss1 mRNA was detected in the hypothalamus of pigs and sheep. It also increases, but earlier than the increase of GnRH. Studies on mice have

Method used

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  • Application of transcription factor CEBP alpha serving as transcription factor of Kiss1 promoter region
  • Application of transcription factor CEBP alpha serving as transcription factor of Kiss1 promoter region
  • Application of transcription factor CEBP alpha serving as transcription factor of Kiss1 promoter region

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 The cultivation of ovarian granulosa cells

[0046] (1) Collect the ovaries at the slaughterhouse, put them in a thermos bottle at 37°C with PBS or normal saline (containing 1% double antibody), and quickly transport them back to the laboratory;

[0047] (2) After washing the collected ovaries with preheated PBS (containing 1% double antibody) for 3 times in a sterile culture room, they were quickly transferred to the ultra-clean workbench; a 1mL sterile disposable syringe was inserted shallowly into the cavity of the ovary Absorb follicular fluid from follicles;

[0048] (3) Place the aspirated follicular fluid in a 15 mL centrifuge tube containing an appropriate amount of DMEM, and centrifuge at 1000 rpm for 6 min at room temperature;

[0049] (4) Discard the supernatant, resuspend and centrifuge in DMEM, and wash the cells twice; prepare DMEM complete medium: 89% DMEM+10% FBS+1% double antibody;

[0050] (5) Aspirate the cell resuspension and complete ...

Embodiment 2

[0052] Example 2 Inoculation and transfection of ovarian granulosa cells

[0053] (1) The granulosa cells grow to about 90%, discard the medium, and wash 3 times with preheated PBS containing 1% double antibodies (the double antibodies are penicillin and streptomycin);

[0054] (2) Add trypsin for digestion, put it in the incubator for about 3 minutes, observe under the microscope until most of the cells float, immediately add the same amount of stop solution to stop the digestion;

[0055] (3) DMEM was washed twice, and centrifuged at 1000rpm for 5min during this period;

[0056] (4) Gently resuspend the cell pellet with complete medium, evenly distribute into each well, supplement the volume with complete medium, shake gently, and culture in the incubator;

[0057] (5) About 24 hours, observe the state of granulosa cells, and prepare for transfection when the confluence of the cells reaches about 80%;

[0058] (6) transfection method is by Invitrogen company's 3000 kit i...

Embodiment 3

[0061] Example 3 qRT-PCR

[0062] The qRT-PCR detection of genes in the present invention uses SYBR SYBR Green qPCRMix kits from Thermo Company, USA. 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:

[0063] 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﹚〉}

[0064] Wherein, GAPDH is used as an internal reference for gene detection, and the qRT-PCR primers used in the present invention are:

[0065] qRT-PCR-Kiss1 Forward: 5′-AACCAGCATCTTCTCACCAGG-3′;

[0066] Reverse: 5'-CTTTCTCTCCGCACAACGC-3';

[0067] qRT-PCR-GAPDH Forward: 5′-TCCCGCCAACATCAAAT-3′;

[0068] Reverse: 5′-CACGCCCATCACAAAACAT-3′;

[0069] qRT-PCR-CEBPα Forward: 5′-CTGAGGTCTGCCAGAAGC-3′;

[0070] Reverse: 5′-AACAGAAGAAGGAAGGGAGT-3′;

[0071] The total RNA of cells was extracted according to the TRIzol operation manual of Takara Company, and the specific ext...

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Abstract

The invention discloses application of a transcription factor CEBP alpha serving as a transcription factor of a Kiss1 promoter region, and belongs to the technical fields of gene engineering and cellengineering. According to the invention, the transcription factor CEBP alpha is taken as a research object, a potentially combined transcription factor CEBP alpha is predicated through bioinformatics,and the influence on the activity of the Kiss1 promoter region by the transcription factor CEBP in ovarian granulosa cell is researched, so as to achieve research on expression regulation of gene inthe ovarian granulosa cell. According to the invention, expression regulation to the Kiss1 gene by the transcription factor CEBP in porcine ovarian granulosa cell and research on functions of the transcription factor CEBP in the ovarian granulosa cell are verified for the first time. The research is rich in experiment content, and results are complete and accurate.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and cell engineering, and specifically relates to the application of transcription factor CEBPα as a transcription factor in the Kiss1 promoter region during the development of sow ovary follicles. Background technique [0002] The growth and development status of ovarian follicles directly determines the reproductive performance of sows, and the function of granulosa cells affects the growth and atresia of follicles. Studies have found that Kiss1 can promote the development of follicles in ovarian tissue and affect the function of granulosa cells. [0003] The ovary is the reproductive organ of female animals, which can provide sex hormones for female animals and provide a place for egg maturation. Studies have shown that FSH and LH secreted by the hypothalamus-pituitary-gonad axis can act on the ovary to promote the growth of ovarian follicles and oocytes. Development, FSH can promot...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/12C12N5/10
CPCC07K14/47C12N15/113C12N15/85C12N2310/141
Inventor 辛晓萍袁晓龙李加琪张哲何颖婷
Owner SOUTH CHINA AGRI UNIV
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