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A miRNA barrier technique

A technology of barrier and technology, applied in the field of biotechnology, can solve the problem of miRNA being negligible, and achieve the effect of stable and high safety of imported genes

Inactive Publication Date: 2007-10-17
HARBIN MEDICAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the current methods for finding and screening miRNA target sites are relatively mature, and more than a thousand miRNAs have been discovered, so far there are very few miRNAs whose functions have been confirmed.

Method used

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  • A miRNA barrier technique
  • A miRNA barrier technique
  • A miRNA barrier technique

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specific Embodiment approach 1

[0012] Specific embodiment 1: The miRNA barrier technology in this embodiment is implemented according to the following steps: (1) Determination of the target gene sequence combined with miRNA in the 3' non-coding region (3'UTR) of the target mRNA; (2) Synthesis and steps (1) The specific antisense oligomeric barrier sequence tightly combined with the measured target gene sequence; (3) The specific antisense oligomeric barrier sequence is transfected into the cell and combined with the target mRNA to form a diploid, that is, the barrier target miRNA to mRNA; 5 nucleotides or deoxynucleotides at the two ends of the specific antisense oligomeric barrier sequence molecule in the step (two) are locked, and the ribose ring is located at the subunit of the 2' oxygen atom and the 4' carbon atom Methyl bridging.

[0013] In the present invention, after the specific antisense oligomeric barrier sequence is introduced into the targeted cells, the action of the endogenous miRNA is blocke...

specific Embodiment approach 2

[0014] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the specific antisense oligomeric barrier sequence in step (2) is 22 nt. Other steps and selected parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0015] Specific embodiment three: the difference between this embodiment and specific embodiment one is: in step (3), calcium phosphate-DNA co-precipitation method, liposome-mediated DNA transfection method or electric shock method are used to transduce specific antisense Oligomeric barrier sequence. Other steps and selected parameters are the same as those in Embodiment 1.

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Abstract

The present invention relates to miRNA barrier technology, belonging to biological technology. The miRNA technology can be implemented according to the following steps: 1) determining the target gene sequence of combining mRNA 3' UTR and miRNA, 2) synthesizing specific anti-sense oligo-barrier sequence, 3) transducing specific anti-sense oligo-barrier sequence into cell and combining target mRNA to form diploid, which can barrier the target miRNA of mRNA. 5 nucleotides or deoxyadenylic acids positioned at two ends of the specific anti-sense oligo-barrier sequence of the step 2) are locked while ribose ring is bridge-linked by 2' oxygen atom and 4' carbon atom. The inventive method has property of good transduced gene stability, high expression. The present invention proves that it is practical for interfering miRNA function using gene specific mode and provides novel measure for researching miRNA function which enable the selective gene therapy strategy and lay foundation to miRNA gene-specific use.

Description

technical field [0001] The present invention relates to a kind of biotechnology. Background technique [0002] miRNAs are a class of non-coding single-stranded RNA molecules with a length of 21-25 nt, which widely exist in plants and cells from nematodes to humans. MicroRNA (microRNA, miRNA) is a molecule that exists widely and fine-tunes gene expression. The miRNA gene accounts for about 1% of the human genome. It is a class of molecules that are highly conserved in evolution and play an important regulatory role in life. . Most of the target genes of miRNA are genes involved in transcription, signal transduction, and tumorigenesis. Although the role of miRNA is also specific, especially the 2-8 bases at the 5′ end, which specifically target its target gene, but Different from siRNA (smallinference RNA), the specificity of miRNA is not so strong. In organisms, one miRNA often acts on multiple target genes or multiple miRNAs regulate one target gene. [0003] At present, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09
Inventor 杨宝峰王志国吕延杰王宁张莹
Owner HARBIN MEDICAL UNIVERSITY
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