An Approach to Genomewide Screens of Expressed Small Interfering RNAs in Mammalian Cells

To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and hum...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2004-01, Vol.101 (1), p.135-140
Main Authors: Zheng, Lianxing, Liu, Jun, Batalov, Sergei, Zhou, Demin, Orth, Anthony, Ding, Sheng, Schultz, Peter G.
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Biological Sciences
Cell Line
Cellular biology
Gene Expression
Gene Silencing
Genes
Genes, Reporter
Genetic screening
Genetic Vectors
Genomic Library
Genomics
HEK293 cells
HeLa Cells
Humans
Lamins
Libraries
Luciferases - genetics
Mammals
Mice
NF-kappa B - genetics
NF-kappa B - metabolism
Plasmids - genetics
Polymerase chain reaction
Promoter Regions, Genetic
Ribonucleic acid
RNA
RNA Polymerase III - genetics
RNA, Small Interfering - genetics
Signal Transduction
Small interfering RNA
Transfection
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Summary:To facilitate the construction of large genomewide libraries of small interfering RNAs (siRNAs), we have developed a dual promoter system (pDual) in which a synthetic DNA encoding a gene-specific siRNA sequence is inserted between two different opposing polymerase III promoters, the mouse U6 and human H1 promoters. Upon transfection into mammalian cells, the sense and antisense strands of the duplex are transcribed by these two opposing promoters from the same template, resulting in a siRNA duplex with a uridine overhang on each 3′ terminus. A single-step PCR protocol has been developed by using this dual promoter system that allows the production of siRNA expression cassettes in a high-throughput manner. We have shown that siRNAs transcribed by either the dual promoter vector or siRNA expression cassettes can induce strong and gene-specific suppression of both endogenous genes and ectopically expressed genes in mammalian cells. Furthermore, we have constructed an arrayed siRNA expression cassette library that targets >8,000 genes with two siRNA sequences per gene. A high-throughput screen of this library has revealed both known and unique genes involved in the NF-κB signaling pathway.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2136685100