A large-scale RNAi screen in human cells identifies new components of the p53 pathway.
RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular signalling pathways. In mammalian cells, such screens have been hampered by a lack of suitable tools that can be used on a large scale. We and others have recently developed expression vectors to direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression. Here we report the construction of a set of retroviral vectors encoding 23,742 distinct shRNAs, which target 7,914 different human genes for suppression. We use this RNAi library in human cells to identify one known and five new modulators of p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest. Furthermore, we describe siRNA bar-code screens to rapidly identify individual siRNA vectors associated with a specific phenotype. These new tools will greatly facilitate large-scale loss-of-function genetic screens in mammalian cells.
Berns
Katrien
K
Division of Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Hijmans
E Marielle
EM
Mullenders
Jasper
J
Brummelkamp
Thijn R
TR
Velds
Arno
A
Heimerikx
Mike
M
Kerkhoven
Ron M
RM
Madiredjo
Mandy
M
Nijkamp
Wouter
W
Weigelt
Britta
B
Agami
Reuven
R
Ge
Wei
W
Cavet
Guy
G
Linsley
Peter S
PS
Beijersbergen
Roderick L
RL
Bernards
René
R
D002455:Cell Division; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005347:Fibroblasts; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D025803:Tumor Suppressor Protein p14ARF / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
C493956:CDKN1A protein, human; C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D034741:RNA, Small Interfering; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53