A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen.
To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.
Moffat
Jason
J
Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA.
Grueneberg
Dorre A
DA
Yang
Xiaoping
X
Kim
So Young
SY
Kloepfer
Angela M
AM
Hinkle
Gregory
G
Piqani
Bruno
B
Eisenhaure
Thomas M
TM
Luo
Biao
B
Grenier
Jennifer K
JK
Carpenter
Anne E
AE
Foo
Shi Yin
SY
Stewart
Sheila A
SA
Stockwell
Brent R
BR
Hacohen
Nir
N
Hahn
William C
WC
Lander
Eric S
ES
Sabatini
David M
DM
Root
David E
DE
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D007990:Libraries; D051379:Mice; D046228:Microarray Analysis; D034741:RNA, Small Interfering / Q000235:genetics*
D018797:Cell Cycle Proteins; D034741:RNA, Small Interfering