A resource for large-scale RNA-interference-based screens in mammals.
Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.
Paddison
Patrick J
PJ
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Silva
Jose M
JM
Conklin
Douglas S
DS
Schlabach
Mike
M
Li
Mamie
M
Aruleba
Shola
S
Balija
Vivekanand
V
O'Shaughnessy
Andy
A
Gnoj
Lidia
L
Scobie
Kim
K
Chang
Kenneth
K
Westbrook
Thomas
T
Cleary
Michele
M
Sachidanandam
Ravi
R
McCombie
W Richard
WR
Elledge
Stephen J
SJ
Hannon
Gregory J
GJ
D000818:Animals; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D005796:Genes / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D046988:Proteasome Endopeptidase Complex; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D034741:RNA, Small Interfering; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex