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 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 >8000 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-kappaB signaling pathway.
Zheng
Lianxing
L
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Liu
Jun
J
Batalov
Sergei
S
Zhou
Demin
D
Orth
Anthony
A
Ding
Sheng
S
Schultz
Peter G
PG
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors; D015698:Genomic Library; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D015398:Signal Transduction; D014162:Transfection
D016328:NF-kappa B; D034741:RNA, Small Interfering; D008156:Luciferases; D012320:RNA Polymerase III