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ShRNA (short hairpin ribonucleic acid) suppressing IRS1 (insulin receptor substrate 1) gene expression and application

A gene expression and gene technology, applied in the field of genetic engineering, can solve the problems of short lifespan of mice, inability to be used as a long-term disease research target, and inability of mice to simulate human diseases well.

Inactive Publication Date: 2014-02-19
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, mouse models still have many limitations. Mice cannot simulate human diseases well at the genetic level and phenotype, and mice have a short lifespan, so they cannot be used as research targets for long-term diseases.

Method used

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  • ShRNA (short hairpin ribonucleic acid) suppressing IRS1 (insulin receptor substrate 1) gene expression and application
  • ShRNA (short hairpin ribonucleic acid) suppressing IRS1 (insulin receptor substrate 1) gene expression and application
  • ShRNA (short hairpin ribonucleic acid) suppressing IRS1 (insulin receptor substrate 1) gene expression and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of porcine IRS gene and its expression in various tissues

[0041] (1) Cloning of porcine IRS1 gene

[0042] According to the known porcine IRS1 gene sequence, 4 pairs of primers (Table 1) were designed, and the full-length porcine IRS1 gene was cloned by overlapping PCR method. Among them, the 1st, 2nd, and 4th pair of primers are exon regions, and do not span introns, and PCR amplification is performed directly using the genome of Bama pigs as a template. Liver cDNA was used as a template for PCR amplification, and the total system was 50ul. The PCR reaction system includes: LA Taq 1ul, 10×LA Taq Buffer 5ul, dNTPs 4ul, template 1ul, Forward Primer 2ul, Reverse Primer 2ul, dH2O 35ul. The PCR reaction parameters were: pre-denaturation at 94°C for 5min; denaturation at 94°C for 30s, annealing at 60°C for 30s, extension at 72°C for 2min, 35 cycles; final extension at 72°C for 10min. The results of PCR products were observed in 1% agarose gel electrophoresis, an...

Embodiment 2

[0052] Construction of p-Genesil-shIRS1 / shIRS2 interference vector

[0053] According to the porcine IRS1 and IRS2 sequences obtained by cloning, four pairs of interference fragments were designed through the Ambion website (Table 2), and single-stranded shRNA interference fragments with BamH I and Hind III restriction sites were synthesized, and after renaturation into double strands, ligation to the p-Genesil 1.0 vector linearized with BamHI and HindIII. After the sequencing was correct, the successfully constructed vectors were p-Genesil-shIRS1-1-4 and p-Genesil-shIRS2-1-4.

[0054] shIRS1-1 and shIRS2-1 are effective interference fragments screened out respectively, re-synthesized single-stranded shIRS2 interference fragments with Nhe I and Xho I restriction sites, annealed into double strands, and connected to the effective screening fragments Nhe I and Xho I linearized p-Genesil-shIRS1 vector. Afterwards, the hU6 promoter was amplified by PCR, with SnaB Ⅰ and Nhe Ⅰ res...

Embodiment 3

[0056] Cultivation of Porcine Liver Primary Cells and Detection of Interfering Carrier Effect

[0057] (1) Culture of primary porcine liver cells

[0058] Take 1d Bama pig liver tissue about 0.5cm 3Place it in physiological saline containing double antibodies, wash the tissue, peel off the mesangium on the surface of the liver, remove blood and other sundries, and wash until the liver tissue is bright red, and the lotion has no obvious sundries. After transferring the tissue to a Petri dish, cut it up with surgical scissors. Add 1ml of collagenase and place in a 37°C incubator for digestion for 5min. Add culture medium to terminate. The tissue was blown away, and the mixture was added to a centrifuge tube for centrifugation at 1200 rpm for 3 min. After centrifugation, discard the supernatant and add 3ml culture medium to resuspend. Inoculate into three large dishes and transfer to a 37°C incubator for cultivation. Observe that the cells have tissue pieces attached to the...

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Abstract

The invention discloses shRNA (short hairpin ribonucleic acid) suppressing IRS1 (insulin receptor substrate 1) gene expression and an application, and belongs to the technical field of gene engineering. A nucleotide sequence of the shRNA is represented by SEQ ID NO.1. The invention further discloses a shRNA interference vector capable of lowering swine-origin IRS1 and IRS2 gene expression simultaneously through connection with a plasmid vector comprising IRS2 genes. Results of swine liver cell IRS gene expression tests indicate that IRS1 genes and the IRS2 genes of swine are lowered by 78% and 64% by the shRNA interference vector respectively. Meanwhile, lowering of the IRS gene expression level affects glucolipid metabolism of swine liver cells, so that the blood sugar concentration of the swine liver cells is increased. According to the shRNA, a foundation is laid for establishment of a type 2 diabetes mellitus swine model, and helpful exploration for increase of the swine blood sugar concentration is conducted.

Description

technical field [0001] The invention relates to a shRNA for inhibiting IRS1 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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Patent Type & Authority Applications(China)
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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