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Small interfering RNAs, short hairpin RNAs, vectors and applications targeting mammalian r-spondin1 gene targets

A mammalian, small-interference technology, applied in the direction of DNA/RNA fragments, vectors, nucleic acid vectors, etc., can solve the problems of short-term inhibition of target gene expression, non-persistent expression of siRNA, low transfection efficiency, etc., to promote the recovery process. Effect

Active Publication Date: 2017-05-03
JIAXING NO 1 HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Initially, RNA interference sampling was the method of synthesizing siRNA in vitro, but its application was limited by the disadvantages of low transfection efficiency, persistent expression of siRNA transferred into cells, and short-term inhibition of target gene expression.

Method used

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  • Small interfering RNAs, short hairpin RNAs, vectors and applications targeting mammalian r-spondin1 gene targets
  • Small interfering RNAs, short hairpin RNAs, vectors and applications targeting mammalian r-spondin1 gene targets
  • Small interfering RNAs, short hairpin RNAs, vectors and applications targeting mammalian r-spondin1 gene targets

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 siRNA sequence design

[0032] Apply the principles of siRNA design (Petri S, siRNA design principles and off-target effects. Methods Mol Biol. 2013; 986:59-71) to screen siRNA sequences: (1) start from the AUG start codon of the transcript (mRNA), search for "AA" double-linked sequence, and write down the 19-23 nucleotide sequence at its 3' end as the candidate target site of siRNA; (2) the GC content of the siRNA sequence is between 45% and 55%; (3) the length 19-23 bp; (4) no inverted repeat sequence; (5) no more than 9 consecutive GC sequences; (6) the 5' end of the sense strand is preferably G / C; (7) antisense The 5' end of the strand is preferably A / U; (8) the 15-19 bases of the sense strand preferably contain more than 3 A / U; (9) the 5' end of the antisense strand is best among the 7 bases There are more than 5 A / U.

[0033] Use Blast (www.ncbi.nlm.nig.gov / Blast) to perform homology analysis on the candidate sequence and the genome database to ensure...

Embodiment 2

[0037] Example 2 shRNA design and synthesis

[0038] Apply the shRNA design principles (Sun G, Molecular Properties, Functional Mechanisms, and Applications of Sliced ​​siRNA. Mol Ther Nucleic Acids. 2015 Jan 20; 4:e221.), according to the above siRNA-R-Spondin1 sequence, design the shRNA sequence: (1) The two complementary oligonucleotides must have restriction enzyme sites at both ends; (2) a C is immediately below the enzyme site to ensure transcription; (3) the shRNA target sequence starts with a G base, To ensure RNA polymerase transcription; (3) The loop adapter sequence inserted by shRNA should be close to the middle of the oligonucleotide, and 5`-TCAAGAG-3` is the most effective; (4) shRNA can only have a specific and unique loop structure;( 5) Insert 5-6 Ts at the end of the shRNA fragment to ensure that RNA polymerase III terminates transcription; (6) G / C content is 40% to 50%; (7) shRNA sequence should avoid more than three consecutive G / C / A / T appears. The obt...

Embodiment 3

[0043] Example 3 Plasmid vector construction

[0044] The above shRNA-R-Spondin1 was synthesized into an oligo DNA single-stranded fragment and annealed to form a double-stranded DNA, which was connected to the pGCL-GFP vector (Shanghai Jikai) to construct the plasmid vector pGCL-GFP-shRNA-R-Spondin1 (see figure 1 ). The plasmid vector will transcribe shRNA in the cell, and produce siRNA-R-Spondin1 targeting the R-Spondin1 gene target after intracellular modification. The annealing and connection process is as follows:

[0045] 1. Annealing into double-stranded DNA

[0046] 1) Establish the following annealing reaction system (room temperature) in a 0.5ml sterile centrifuge tube:

[0047]

[0048] 2) Incubate at 95°C for 4 minutes and at 70°C for 10 minutes;

[0049] 3) Take out the centrifuge tube, place it at room temperature for 5-10 minutes, and cool to room temperature;

[0050] 4) Briefly centrifuge and mix well.

[0051] 2. Ligation of double-stranded DNA to...

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Abstract

The invention discloses a small-interfering RNA, a short hairpin RNA and a carrier for a mammal R-Spondin1 gene target as well as application thereof. The small-interfering RNA contains a positive-sense strand and an antisense strand. The short hairpin RNA is synthetized on the basis of the small-interfering RNA by virtue of a chemical synthesis method, and can be further connected with a virus expression vector or a non-virus expression vector so as to form the carrier for the mammal R-Spondin1 gene target; the virus expression vector is a lentiviral vector or an adenovirus vector, the non-virus expression vector is a plasmid vector; the carrier of the short hairpin RNA can be used for preparing gene therapy medicine for hepatic fibrosis. The RNA interference fragment designed for the R-Spondin1 gene target is capable of promoting static state back-formation or apoptosis of activated hepatic stellate cells and thus effectively promoting a hepatic fibrosis recovery process.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a small interfering RNA, a short hairpin RNA, a carrier and an application for a mammalian R-Spondin1 gene target. Background technique [0002] Hepatic fibrosis is the wound healing response of the liver to chronic liver injury caused by various reasons, resulting in a large amount of fibrous tissue hyperplasia and precipitation in the hepatic lobule and portal area, and the pathological feature is the synthesis of various components of the extracellular matrix mainly composed of collagen Increased, relatively insufficient degradation, but did not form interlobular septa, if further developed into cirrhosis. Liver fibrosis is a reversible process, and the prevention and early intervention of liver fibrosis are the best measures to stabilize the disease and prevent the development of liver fibrosis to cirrhosis and liver cancer. [0003] Hepatic stellate cells are the main cells tha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85A61K48/00A61K31/713A61P1/16
CPCC12N15/1136C12N15/85C12N2310/141C12N2310/531C12N2320/30C12N2330/30C12N2800/107
Inventor 虞玲华殷新光
Owner JIAXING NO 1 HOSPITAL
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