Interference RNA miniplasmid of hypoxia induction factor

A hypoxia-inducible factor and small interference technology, applied in the field of genetic engineering, can solve the problems of poor RNA chain stability, unfavorable transfection efficiency, easy degradation, etc., and achieve stable and high-efficiency expression

Inactive Publication Date: 2006-09-13
RENJI HOSPITAL ATTACHED TO SHANGHAI NO 2 MEDICALUNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, the selection of siRNA fragments is often non-specific, and the RNA chain has poor stability and is easy to degrade, which is not conducive to further improving transfection efficiency.
[0006] Compared with the prese

Method used

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  • Interference RNA miniplasmid of hypoxia induction factor
  • Interference RNA miniplasmid of hypoxia induction factor
  • Interference RNA miniplasmid of hypoxia induction factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of siRNA-HIF-1α

[0027] Design of target gene HIF-1α siRNA sequence

[0028] Open the siRNA design tool http: / / www.ambion.com / techlib / misc / siRNA_finder.html, the CDS of human hypoxia-inducible factor 1α (HIF-1α) gene (the CDS (29-2509) of HIF-1α sees SEQID NO: 4) Paste in the input box, select end with TT, and get a series of output sequences of 21 bases (nt) after submission, select 1 to 2 sequences, and confirm that there is no significant similarity with other genes by Blast test , converting the output sequence into two insertion sequences, the sense strand and the antisense strand.

[0029] The selected sequence is as follows:

[0030] The obtained siRNA-upstream sequence is (SEQ ID NO: 1):

[0031] 5′-TGTGAGTTCGCATCTTGATTTCAAGAGAATCAAGATGCGAACTCACATTTTTT-3′

[0032] Among them, TGTGAGTTCGCATCTTGAT is the obtained upstream segment; TTCAAGAGA is a specific loop ring; ATCAAGATGCGAACTCACA is the palindrome structure of the upstream segment. ...

Embodiment 2

[0045] Example 2 Detection of siRNA-HIF-1α

[0046] By electroporation, exogenous siRNA-HIF-1α was transfected into endothelial progenitor cells, and compared with the non-transfected siRNA group as follows.

[0047] 1. Genetic testing

[0048] After 3 days of culture, the cells were transfected with plasmids, incubated in normoxia and hypoxia for 20 hours, and then RT-PCR and β-actin were quantified. The electrophoresis results are shown in Figure 2, in which, lane 1 is a 100bp marker; lanes 2, 4, and 6 are normoxia; lanes 3, 5, and 7 are hypoxia; lanes 2-3 are not transfected cells; lanes 4-5 are pEGFP empty Plasmid-transfected cells; lanes 6-7 are SiRNA-HIF-1α-transfected cells. A is HIF-1α electrophoresis 20 hours after transfection, B is VEGF electrophoresis 20 hours after transfection. The results showed that: HIF1αCDNA exists in both normoxia and hypoxia, and its expression has nothing to do with oxygen concentration. SiRNA interference inhibits HIF1α; VEGF expressio...

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Abstract

The invention discloses a low oxygen induction factor low interruption RNA SiRNA-HIF-1 alpha particle. The particle could be high efficiently expressed in cell and could strongly restrict the expression of independent gene in mammal cell.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to a genetic engineering plasmid. Furthermore, the invention relates to a hypoxia-inducible factor small interfering RNA plasmid capable of stably and efficiently expressing in cells. Background technique [0002] RNA interference (or called RNA interference, RNA interference, RNAi) refers to the process of degrading the specific mRNA paired with it induced by double-stranded RNA (dsRNA) in cells. In some biological systems, a small number of copies of dsRNA can lead to the degradation of all homologous genes in the cell. This phenomenon is conserved and widespread during evolution, and many genes play important roles in this process. This phenomenon is widespread in nematodes, insects, mammals, plants and fungi. [0003] When the long-chain dsRNA enters the cell, it is recognized by a Dicer nucleolytic enzyme, cut into a small interfering RNA (SiRNA) of 21-25 bases, and the double...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66
Inventor 王长谦姜萌
Owner RENJI HOSPITAL ATTACHED TO SHANGHAI NO 2 MEDICALUNIV
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