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.
Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA.
Jason
J
Moffat
Dorre A
DA
Grueneberg
Xiaoping
X
Yang
So Young
SY
Kim
Angela M
AM
Kloepfer
Gregory
G
Hinkle
Bruno
B
Piqani
Thomas M
TM
Eisenhaure
Biao
B
Luo
Jennifer K
JK
Grenier
Anne E
AE
Carpenter
Shi Yin
SY
Foo
Sheila A
SA
Stewart
Brent R
BR
Stockwell
Nir
N
Hacohen
William C
WC
Hahn
Eric S
ES
Lander
David M
DM
Sabatini
David E
DE
Root
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