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siRNA for inhibiting gene expression of mice gonadotropin inhibitory hormone and application of siRNA

A gonadotropin and inhibitory hormone technology, applied in the field of molecular biology, can solve the problem that gonadotropin inhibitory hormone has not been reported, and achieve the effect of high silencing effect and reduced apoptosis of granulosa cells

Pending Publication Date: 2017-07-18
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] As a tool for gene silencing, siRNA has been widely used in many research fields, but siRNA for gonadotropin-inhibiting hormone in mice has not been reported yet

Method used

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  • siRNA for inhibiting gene expression of mice gonadotropin inhibitory hormone and application of siRNA
  • siRNA for inhibiting gene expression of mice gonadotropin inhibitory hormone and application of siRNA
  • siRNA for inhibiting gene expression of mice gonadotropin inhibitory hormone and application of siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Verification of GnIH expression in mouse ovarian granulosa cells

[0041] 1. According to Invitrogen's TRIzoL Reagent kit, extract RNA from mouse ovarian granulosa cells (cells are kept in our laboratory). All the equipment used is soaked in 0.1% DEPC water overnight and then autoclaved. The test tubes used were all RNA-free EP tubes. After the collected cells were dissolved by Trizol, chloroform was extracted and precipitated with isopropanol, the RNA was dissolved with 0.1% DEPC water, and the first strand of cDNA was synthesized using this as a template.

[0042] 2. Using the first strand of cDNA as a template, design PCR primers: mouse gonadotropin inhibitory hormone (GnIH) sequence (GeneBank accession number NM021892), upstream primer sequence: ATGCCTATCACATTGCTG (SEQ ID NO: 13), downstream primer sequence: CAAGGTGAAGACGGAAGC (SEQ ID NO: 14); GnIH receptor GnIH-R (GeneBank accession number NM13392), upstream primer sequence: CAGAAAGCAGGTGGATGG (SEQ ID NO: 15),...

Embodiment 2

[0043] Example 2 Construction of pLV-GnIH-shRNA lentiviral interference plasmid

[0044] 1. According to the mRNA sequence information of mouse gonadotropin inhibitory hormone, according to the siRNA design method and target sequence screening, four siRNA sequences are designed, which are reverse complementary to the mRNA of gonadotropin inhibitory hormone gene, named GnIH-1, GnIH-2, GnIH-3, GnIH-4, the sense strand sequences are: GnIH-1, as shown in SEQ ID NO:1; GnIH-2, as shown in SEQ ID NO: 2; GnIH-3, as SEQ ID NO: 3; GnIH-4, as shown in SEQ ID NO: 4.

[0045] 2. According to the target sequence, design and construct the DNA sequence required for the siRNA vector that inhibits the expression of mouse gonadotropin inhibiting hormone gene: GnIHshRNA template, introduce BamH I and EcoR I restriction sites at both ends, the forward sequence and the reverse sequence Insert the loop structure of CTCGAG in between, and the 3'end of the sense chain sequence will stop the reaction with ...

Embodiment 3

[0055] Example 3 Lentivirus packaging and titer determination

[0056] 1. Transfection of lentiviral plasmid into 293FT cells. The pLV-GnIH-shRNA1, pLV-GnIH-shRNA2, pLV-GnIH-shRNA3, pLV-GnIH-shRNA4 and the empty plasmid pYr-Lvsh (named pLV-shNC) and the packaging plasmid (pLP1, pLP2, pLP / VSVG, see image 3 B) Co-transfect 293FT cells, collect the virus liquid, and measure the titer after virus concentration.

[0057] Liposomal transfection, according to Lipofectamine TM 2000 kit instructions were performed.

[0058] (1) The day before transfection, inoculate cells in a new culture dish. The cells must be completely blown into a single state to ensure uniform distribution. Transfection is performed when the cell confluence reaches 70-80%;

[0059] (2) 2h before transfection, replace the culture medium with fresh serum-free medium;

[0060] (3) Add interfering plasmids (empty plasmids), pLP1, VSVG and pLP2 at the ratios of 10μg, 6.5μg, 3.5μg, and 2.5μg into a centrifuge tube containing...

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Abstract

The invention relates to siRNA for inhibiting the gene expression of mice gonadotropin inhibitory hormone (GnIH). A positive-sense strand of the siRNA has any sequence shown in SEQ ID NO.1 to 4. The invention constructs lentivirus interference plasmid of a coding gene containing the siRNA and carries out an in-vitro cellular level experiment on the lentivirus interference plasmid, a result shows that after being transferred in a cell, the lentivirus interference plasmid is capable of expressing shRNA, and can perform specific inhibition on a GnIH gene, and two siRNA fragments can enable the mRNA expression of the mice ovarian granular cells GnIH to be decreased by 70 percent or above. After the GnIH expression is inhibited, the proliferation of the mice ovarian granular cells and the secretion of estrogen can be significantly increased, and the granular cell apoptosis is inhibited. Therefore, the siRNA fragment and an expression carrier thereof can be used for preparing a preparation for expressing the mice GnIH.

Description

Technical field [0001] The invention relates to the field of molecular biology, in particular to a siRNA fragment for inhibiting the expression of mouse gonadotropin inhibiting hormone gene and its application. Background technique [0002] In the regulation of the secretion of animal gonadal hormones, gonadotropin-inhibitory hormone (GnRH) has always been regarded as the only neuropeptide that regulates the synthesis and secretion of gonadal hormones. It was not until 2000 that the Japanese quail hypothalamus A new RF amide peptide (SIKPSAYLPLRFamide) with a 12 amino acid sequence was isolated from the Chinese Academy of Sciences. Because its role in the regulation of hormone secretion is opposite to GnRH, it is called gonadotropin-inhibitory hormone (GnIH) . In subsequent studies, homologous analogues of GnIH were isolated in different animals. GnIH acts on the pituitary gland or GnRH neurons, and inhibits the synthesis and secretion of follicle stimulating hormone (FSH) and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61K31/7088A61P5/04
CPCC12N15/1136C12N2310/14C12N2310/531
Inventor 王水莲曾杰阳美霞龚金秋唐姣美
Owner HUNAN AGRICULTURAL UNIV
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