A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells

Stegmeier, F., Hu, G., Rickles, R. J., Hannon, G. J., Elledge, S. J. (September 2005) A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proceedings of the National Academy of Sciences of the United States of America, 102 (37). pp. 13212-13217. ISSN 0027-8424

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URL: https://www.ncbi.nlm.nih.gov/pubmed/16141338
DOI: 10.1073/pnas.0506306102

Abstract

The advent of RNA interference has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. The expression of short hairpin RNA (shRNA) from polymerase III promoters can be encoded in transgenes and used to produce small interfering RNAs that downregulate specific genes. In this study, we show that polymerase II-transcribed shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a microRNA context. Importantly, our shRNA expression system [called PRIME (potent RNA interference using microRNA expression) vectors] allows for the multicistronic cotranscription of a reporter gene, thereby facilitating the tracking of shRNA production in individual cells. Based on this system, we developed a series of lentiviral vectors that display tetracycline-responsive knockdown of gene expression at single copy. The high penetrance of these vectors will facilitate genomewide loss-of-function screens and is an important step toward using bar-coding strategies to follow loss of specific sequences in complex populations.

Item Type: Paper
Uncontrolled Keywords: genetic screen lentivirus MAGIC Gene expression pathway screen
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes > RNA polymerase
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > miRNA
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > miRNA

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > shRNA
CSHL Authors:
Communities: CSHL labs > Hannon lab
Depositing User: CSHL Librarian
Date: September 2005
Date Deposited: 05 Jan 2012 14:33
Last Modified: 11 Jan 2018 17:24
PMCID: PMC1196357
Related URLs:
URI: http://repository.cshl.edu/id/eprint/22714

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