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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.

Pubmed ID: 16564017


  • Moffat J
  • Grueneberg DA
  • Yang X
  • Kim SY
  • Kloepfer AM
  • Hinkle G
  • Piqani B
  • Eisenhaure TM
  • Luo B
  • Grenier JK
  • Carpenter AE
  • Foo SY
  • Stewart SA
  • Stockwell BR
  • Hacohen N
  • Hahn WC
  • Lander ES
  • Sabatini DM
  • Root DE



Publication Data

March 24, 2006

Associated Grants

  • Agency: NCI NIH HHS, Id: CA103866
  • Agency: NCI NIH HHS, Id: P50 CA112962
  • Agency: NCI NIH HHS, Id: R01CA97061

Mesh Terms

  • Animals
  • Cell Cycle Proteins
  • Cells, Cultured
  • Gene Library
  • Genetic Engineering
  • Genetic Vectors
  • Humans
  • Lentivirus
  • Libraries
  • Mice
  • Microarray Analysis
  • RNA, Small Interfering