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Method for the in vitro synthesis of short double stranded RNAs

A technology of RNA polymerase and polymerase, which is applied in the field of synthesizing short double-stranded target-specific RNAs, and can solve the problems that small interfering RNAs cannot be directly applied

Inactive Publication Date: 2005-04-06
JANSSEN PHARMA NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Existing in vitro methods for the synthesis of small single-stranded RNAs of specific length and sequence (Milligan F. et al., 1987, Nucleic Acid Res. (15) 8783-8798), cannot be directly applied to the synthesis of small interfering RNAs
The problem lies in the fact that RNA polymerase tends to transcribe certain nucleotides of the promoter sequence into the transcript

Method used

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  • Method for the in vitro synthesis of short double stranded RNAs
  • Method for the in vitro synthesis of short double stranded RNAs
  • Method for the in vitro synthesis of short double stranded RNAs

Examples

Experimental program
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Effect test

Embodiment 1

[0088] Example 1: EGFP and GL3-specific short dsRNAs transcribed in vitro, in human

[0089] Inducible RNA transfection in cell-like cells

[0090] Materials and methods

[0091] plasmid construct

[0092] Luciferase+ was expressed from the plasmid pGL3-control (Promega). EGFP is expressed by EGFP / pcDNA5-FRT, which includes the EGFP gene from pEGFP (Clontech), which is directionally linked to the HindIII and NotI sites of the mammalian expression vector pcDNA5 / FRT (Invitrogen).

[0093] In vitro transcription and hybridization of siRNAs

[0094] Hybridize the oligomer template strand to the sense T7 promoter sequence (5' TAATACGACTCACTATAGG) in 10 mM Tris-HCl pH 9.0, 100 mM NaCl, 1 mM EDTA, including boiling for 2 minutes and slowly cooling over 2-3 hours to room temperature.

[0095] Transcription is using MEGAshortscript TM T7 kit (Ambion), according to the manufacturer's instructions. siRNA was purified on G-25 spin columns. Phenol:chloroform:isoamyl...

Embodiment 2

[0116] Example 2: Mouse Insr-specific short dsRNAs transcribed in vitro, knocking out Balb / C

[0117] Insr in mouse liver

[0118] Male Balb / C mice (approximately 25 g) (standard housing, free access to food / water) received a tail vein injection with 2.3 ml of saline, or 40 μg of the mouse insulin receptor-targeted (NCBI deposit number NM-010568; 2536 -2556 bp) of siRNA prepared by the truncated T7 promoter method of in vitro transcription, while using 800 U RNase inhibitor.

[0119] The injections were performed as quickly as possible (8-10 seconds) and 2 control and 2 siRNA-treated mice were sacrificed at 24, 48 and 72 hours. The liver was quickly removed, weighed, and frozen in dry ice / isopropanol. Total RNA was extracted using crushed frozen tissue and the RNEasy Maxi kit (Qiagen).

[0120] After first-strand cDNA synthesis, insulin receptor mRNA was analyzed by Q-PCR using a Smart cycler (primers: F 3526-3548, R 3744-3768), and the results were also ...

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Abstract

The present invention relates to the field of synthesis of short double-stranded RNAs. An in vitro transcription method using bacteriophage polymerases and target sequence-specific single-stranded DNA oligonucleotides as templates is disclosed. The present invention finds particularly advantageous use in the synthesis of short interfering RNAs (siRNAs) that have been shown to function as key intermediates in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates and vertebrate systems.

Description

[0001] The present invention relates to the field of synthesis of short double-stranded target-specific RNAs. In vitro transcription methods utilizing RNA polymerase and using target sequence-specific DNA oligonucleotides as templates are disclosed. The invention is particularly applicable to the synthesis of short interfering RNAs (siRNAs), which have been shown to cause sequence-specific RNA degradation during post-transcriptional gene silencing in plants, as well as in RNA interference in invertebrate and vertebrate systems. key intermediates. Background of the invention [0002] RNA silencing is an important type of gene regulation based on the sequence-specific targeting and degradation of RNA. RNA silencing was first identified in transgenic plants, where it is called co-suppression or post-transcriptional gene silencing (PTGS). Only recently has the sequence-specific RNA degradation process, RNA interference (RNAi), associated with PTGS been identified in ciliates, fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K31/7105A61K38/00A61K48/00A61P35/00C12N15/10C12N15/11C12P19/34C12Q1/68
CPCC12N2310/14C12Q1/6865C12P19/34C12N2310/53C12N15/10A61K38/00C12N15/111C12N2330/30C12N2310/111A61P35/00C12Q2525/143C12N15/11
Inventor M·D·德贝克A·N·哈里斯
Owner JANSSEN PHARMA NV
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