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Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell

a nucleic acid sequence and post-transcriptional technology, applied in the field of identifying nucleic acid sequences that modulate the function of cells, can solve the problems that dsrna can interfere with the expression of genes in mouse embryos, and achieve the effects of high throughput identification, rapid identification, and increased half-life of double stranded rna

Inactive Publication Date: 2015-08-20
ALNYLAM PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for identifying nucleic acid sequences that modulate the function of a cell, gene expression in a cell, or the biological activity of a target polypeptide in a cell. This is useful for identifying the genes involved in cell fate decisions and gene silencing events. The method involves transforming a population of cells with a double stranded RNA expression library, and evaluating the effects of PTGS events on cell function simultaneously. The double stranded RNA used in the library can contain regions of complimentarity to each other, and can be designed to have at least 70, 80, 90, 95, 98, or 100% complimentarity. The method can also be used to identify nucleic acid sequences that inhibit the interferon response, which can occur when dsRNA is introduced into cells. Overall, the method allows for high throughput identification of nucleic acid sequences involved in cell fate decisions and gene silencing events.

Problems solved by technology

Furthermore, studies in mice have demonstrated that dsRNA can interfere with the expression of genes in mouse embryos.

Method used

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  • Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell
  • Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell
  • Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell

Examples

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example 1

Design and Delivery of Vectors for Intracellular Synthesis of dsRNA for Library Based Screening Approaches to Nucleic Acid Identification Using PTGS

[0114]PTGS is induced when dsRNA is made intracellularly. The library based screening approaches to nucleic acid identification through PTGS may require that dsRNA reside in certain cellular compartments in order to exert its effect. Therefore, expression plasmids that transcribe dsRNA in the cytoplasm and in the nucleus are utilized. There are two classes of nuclear transcription vectors: one that is designed to express polyadenylated dsRNA (for example, a vector containing an RNA polymerase II promoter and a poly A site) and one that expresses non-adenylated dsRNA (for example, a vector containing an RNA polymerase II promoter and no poly A site, or a vector containing a T7 promoter). Different cellular distributions are predicted for the two species of RNA; both vectors are transcribed in the nucleus, but the ultimate destinations of ...

example 2

Generation of Templates for In Vitro Transcription of dsRNA for Non-Library Based Approaches for Identification of Nucleic Acids Using PTGS

[0130]Nucleic acid sequences that modulate cell function, gene expression in a cell, or the biological activity of a target polypeptide in a cell may also be identified using non-library based approaches involving PTGS. A single known nucleic acid sequence encoding a polypeptide with unknown function or a single nucleic acid fragment of unknown sequence and / or function can be made into a double stranded RNA molecule. This dsRNA is then transfected into a desired cell type and assayed for modulations in cell function, gene expression in the cell, or the biological activity of a target polypeptide in the cell, using methods described herein. A modulation in cell function, gene expression in the cell, or the biological activity of a target polypeptide in the cell identifies the nucleic acid of the dsRNA as a nucleic acid the modulates the specific c...

example 3

In Vitro RNA Transcription and RNA Analysis

[0135]In vitro transcription reactions are carried out using the Riboprobe Kit (Promega Corp.), according to the manufacturer's directions. The template DNA is as described above. Following synthesis, the RNA is treated with RQ1 DNase (Promega Corp.) to remove template DNA. The RNA is then treated with Proteinase K and extracted with phenol-chloroform to remove contaminating RNases. The RNA is ethanol precipitated, washed with 70% ethanol, and resuspended in RNase-free water. Aliquots of RNA are removed for analysis and the RNA solution is flash frozen by incubating in an ethanol-dry ice bath. The RNA is stored at −80° C.

[0136]As an alternative to phenol-chloroform extraction, RNA can be purified in the absence of phenol using standard methods such as those described by Li et al. (WO 00 / 44914, filed Jan. 28, 2000). Alternatively, RNA that is extracted with phenol and / or chloroform can be purified to reduce or eliminate the amount of phenol ...

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Abstract

Described herein are methods for identifying nucleic acid sequences that modulate the function of a cell, the expression of a gene in a cell, or the biological activity of a target polypeptide in a cell. The methods involve the use of double stranded RNA expression libraries, double stranded RNA molecules, and post-transcriptional gene silencing techniques.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit of the filing date of U.S. provisional applications 60 / 265,805, filed Jan. 31, 2001, and 60 / 339,260, filed Oct. 26, 2001.BACKGROUND OF THE INVENTION[0002]The invention relates to methods for identifying nucleic acid sequences that modulate the function of a cell, by the use of post-transcriptional gene silencing.[0003]Double stranded RNA (dsRNA) has been shown to induce sequence-specific gene silencing in a number of different organisms. Gene silencing can occur through various mechanisms, one of which is post-transcriptional gene silencing (PTGS). In post-transcriptional gene silencing, transcription of the target locus is not affected, but the RNA half-life is decreased. The mechanisms by which PTGS occurs are not yet clear. Exogenous dsRNA has been shown to act as a potent inducer of PTGS in nematodes, trypanosomes, and insects. In addition, studies in C. elegans and Drosophila show that a few molecul...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N33/68C12N15/10
CPCC12N15/1096C12Q1/6897C12N15/1086C12Q1/6876C12Q2600/136C12Q2600/16C12Q2600/178G01N33/6866G01N2333/56G01N2333/565G01N2333/57G01N2500/10
Inventor GIORDANO, TONYPACHUK, CATHERINESATISHCHANDRAN, CHANDRASEKHAR
Owner ALNYLAM PHARMA INC
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