shRNA (short hairpin ribonucleic acid) capable of inhibiting IRS (insulin receptor substrate) 2 gene expression and application thereof

A gene expression and product technology, applied in the field of genetic engineering, can solve the problems that can not be used as long-term disease research targets, mice have short lifespan, and mice cannot simulate human diseases well

Inactive Publication Date: 2014-02-26
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, mouse models still have many limitations. Mice cannot simulate human diseases well at the genetic lev...

Method used

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  • shRNA (short hairpin ribonucleic acid) capable of inhibiting IRS (insulin receptor substrate) 2 gene expression and application thereof
  • shRNA (short hairpin ribonucleic acid) capable of inhibiting IRS (insulin receptor substrate) 2 gene expression and application thereof
  • shRNA (short hairpin ribonucleic acid) capable of inhibiting IRS (insulin receptor substrate) 2 gene expression and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Cloning and screening of porcine IRS2 gene

[0023] (1) Cloning of the 3'UTR region of the pig IRS2 gene

[0024] The human IRS2 gene sequence is known. Compare the human IRS2 gene with the pig genome, design primers according to the conserved region sequence, and use 2d Bama pig liver tissue cDNA as a template to clone the conserved sequence of the pig IRS2 gene. PCR Reaction system 20ul: cDNA 0.5ul, primer 2ul, rTaq 0.5ul, 10×PCR Buffer 2.5ul, dNTPs 2ul, dH 2 O17.5ul. The PCR reaction program was: 94°C for 5min; 35 cycles of 94°C for 30s, 59.3°C for 30s, and 72°C for 30s; 72°C for 10min. The results of PCR products were observed in 1% agarose gel electrophoresis, and the target band was recovered from the gel, connected to the PMD-18T cloning vector for sequencing. The mutation-free sequence was obtained for 3' RACE amplification, and the standard operation was performed according to the instructions of the Takara kit.

[0025] The results showed that the obtained...

Embodiment 2

[0027] Construction of p-Genesil-shIRS2 interference vector

[0028] According to the porcine IRS2 sequence obtained by cloning, four pairs of interference fragments were designed through the Ambion website (Table 1), and single-stranded shRNA interference fragments with BamHI and HindIII restriction sites were synthesized, and after renaturation into double strands, they were connected to Ⅰ and HindⅢ linearized p-Genesil1.0 vector. After the sequencing was correct, the successfully constructed vector was p-Genensil-shIRS2-1-4. The effective interference vector screened out is p-Genensil-shIRS2-1.

[0029] Table 1 Interfering fragment sequence of pig IRS2 gene

[0030]

[0031]

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Abstract

The invention discloses shRNA (short hairpin ribonucleic acid) capable of inhibiting IRS (insulin receptor substrate) 2 gene expression and application thereof, belonging to the technical field of gene engineering. The nucleotide sequence of the shRNA is shown in SEQ ID No.1. The invention also provides an interference vector of shRNA capable of inhibiting IRS 2 gene expression. Through cloning the compared and analyzed pig source IRS 2 gene conserved sequence, interference fragments with restriction enzyme cutting site BamH I and Hind III are synthesized, the interference fragments are connected onto a plasmid vector linearized by BamH I and Hind III, and effective interference vectors are screened out. The shRNA and the interference vector thereof can effectively inhibit IRS2 gene expression, affects cell glucolipid metabolism, and lays a foundation for construction of type 2 diabetic pig model.

Description

technical field [0001] The invention relates to a shRNA for inhibiting IRS2 gene expression and its application, belonging to the technical field of genetic engineering. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the phenomenon that when a double-stranded RNA (double stranded RNA, dsRNA) homologous to the endogenous mRNA coding region is introduced into the cell, the mRNA is degraded and gene expression is silenced. It can Inhibits the expression of specific genes in normal organisms. After the exogenous dsRNA enters the cell, the antisense strand of small interfering RNA (siRNA) and a variety of nucleotidases will form a silencing complex (RNA-induced silencing complex) that can bind and cut mRNA. , RISC), which ultimately mediates the process of RNA interference. RNAi can controllably turn off up to 10 genes in a shorter time than the time-consuming gene knockout technology. With its flexible and fast characteristics, RNAi technolo...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/66A01K67/027
Inventor 孔庆然黄天晴刘忠华
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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