Application of FGFR1 genes to granulosa cells of sows

A technology of granulosa cells and genes, applied in the application field of FGFR1 gene in porcine ovary granulosa cells

Active Publication Date: 2019-06-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are no relevant reports on the regulation of the expression

Method used

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  • Application of FGFR1 genes to granulosa cells of sows
  • Application of FGFR1 genes to granulosa cells of sows
  • Application of FGFR1 genes to granulosa cells of sows

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Construction of FGFR1 Gene Overexpression Vector

[0034] BioEdit software analysis found that the CDS region sequence of the FGFR1 gene has no restriction endonuclease sites BamH I and Xba I, but the pcDNA3.1 vector (purchased from Invitrogen, Cat. No. V79020) has BamH I and Xba I restriction sites point. The primer premier 5.0 software designed the primers for the CDS region of the FGFR1 gene, and the upstream and downstream primers were added with BamH I and Xba I restriction site sequences. Using the cDNA of porcine ovary granulosa cells as a template, the target fragment was amplified by PCR, purified and recovered, double enzyme digested, connected to the pcDNA3.1 vector, transformed, screened, sequenced and identified correctly, and then the endotoxin-free plasmid was extracted (a small amount of endotoxin-free plasmid The extraction kit was purchased from Magen Company) and named pcDNA3.1-FGFR1.

Embodiment 2

[0035] The cultivation of embodiment 2 ovarian granulosa cells

[0036] (1) Put the ovarian tissue collected in the slaughterhouse into PBS or normal saline containing 1% double antibody, put it on ice and bring it back to the laboratory quickly;

[0037] (2) Wash the collected ovaries three times with PBS or normal saline (containing 1% double antibody) in a sterile culture room, then quickly transfer them to the ultra-clean workbench, and use a 1 mL sterile disposable syringe to shallowly insert the antral follicles of the ovary absorb follicular fluid;

[0038] (3) Place the aspirated follicle fluid in a centrifuge tube containing an appropriate amount of DMEM, and centrifuge at room temperature for 5 min at 800 rpm;

[0039] (4) Discard the supernatant, then resuspend in DMEM, centrifuge, and wash the cells twice; prepare DMEM complete medium: 89% high-glucose DMEM+10% FBS+1% double antibody;

[0040] (5) Resuspend cells with complete medium, inoculate in 75mL culture fl...

Embodiment 3

[0042] Example 3 Inoculation and transfection of ovarian granulosa cells

[0043] (1) When the confluence of granulosa cells reaches about 90%, the medium is discarded, and the cells are washed 3 times with preheated PBS;

[0044] (2) Add 0.25% trypsin for digestion, put it in the incubator for about 3 minutes, observe under the microscope until most of the cells float up, immediately add an equal amount of stop solution (complete medium) to stop the digestion;

[0045] (3) PBS was washed twice, and centrifuged at 800 rpm for 5 minutes;

[0046] (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;

[0047] (5) About 24 hours, observe the state of granulosa cells, and prepare for transfection when the confluence of the cells reaches about 70-90%;

[0048] (6) transfection method is by Invitrogen company's 3000 kit instructions were carried out; each ...

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Abstract

The invention discloses an application of FGFR1 genes to granulosa cells of sows, and belongs to the technical field of cell engineering and genetic engineering. The FGFR1 genes can be used for detecting the relative expression quantity of FGFR1 mRNA in ovarian tissues of sows of 180 days old (the ovarian is immature ) and sows of 200 days old (the ovarian is mature), and granulosa cells of different size, and the proliferation and apoptosis situation of the granulosa cells after overexpression/interference FGFR1, so as to accumulate materials for the molecule mechanism in the development process of the granulosa cells of the sows. Besides, the results show that the FGFR1 genes participate in promoting proliferation of the granulosa cells, and restraining the apoptosis of the granulosa cells, and further, the results indicate that the FGFR1 genes can participate in development and mature of the granulosa cells of the sows.

Description

technical field [0001] The invention belongs to the technical field of cell engineering and genetic engineering, and specifically relates to an application of FGFR1 gene in porcine ovarian granulosa cells. Background technique [0002] After puberty in mammals, with the growth of the hypothalamus-pituitary-gonad axis and the improvement of secretion function, the ovarian function is basically mature, and the ovarian follicles are gradually matured and ovulated under the stimulation of reproductive hormones, and a periodic process is formed. According to the morphological and structural changes of follicle development, follicles can be roughly divided into three growth stages: primordial follicles, growing follicles, and mature follicles. [0003] Follicles are mainly composed of oocytes, granulosa cells and theca cells. During follicle development, granulosa cells first surround an oocyte in a single-layer flat form to form a primordial follicle, and gradually change from f...

Claims

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Application Information

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IPC IPC(8): C12Q1/6876C12N15/113C12N5/071
Inventor 李加琪李忠慧袁晓龙张哲钟玉宜
Owner SOUTH CHINA AGRI UNIV
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