title: An optimized lentivirus-mediated RNAi screen reveals kinase modulators of kinesin-5 inhibitor sensitivity.
abstract: Abstract: Induction of RNA interference (RNAi) in human cells has enabled comprehensive functional annotation of the human genome via reverse genetic screens. Here we describe an optimized semiautomated method to produce, titrate, and screen large collections of short hairpin RNA (shRNA)-containing lentiviral vectors. We also present results from a pilot lentiviral RNAi screen for kinases whose silencing modulates sensitivity to a mitotic spindle protein kinesin-5 inhibitor (kinesin-5i). Our screen identified three distinct serine/threonine kinase 6 shRNA vectors within our library as enhancers of kinesin-5i-mediated HT29 cell growth inhibition. In contrast, three distinct shRNAs targeting cell division cycle 2/cyclin-dependent kinase 1 resulted in kinesin-5i resistance. These results demonstrate the feasibility of screening with large collections of lentiviral vectors to identify drug enhancers and suppressors.
authors.affiliation: Rosetta Inpharmatics LLC, Merck & Co., Inc., Seattle, WA 98109, USA. richard_klinghoffer@merck.com
authors.forename: Richard A
authors.initials: RA
authors.lastname: Klinghoffer
authors.forename: Brian
authors.initials: B
authors.lastname: Roberts
authors.forename: James
authors.initials: J
authors.lastname: Annis
authors.forename: Jason
authors.initials: J
authors.lastname: Frazier
authors.forename: Patrick
authors.initials: P
authors.lastname: Lewis
authors.forename: Peter S
authors.initials: PS
authors.lastname: Linsley
authors.forename: Michele A
authors.initials: MA
authors.lastname: Cleary
mesh_terms: D001331:Automation; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival; D004353:Drug Evaluation, Preclinical; D020868:Gene Silencing / Q000187:drug effects; D005822:Genetic Vectors; D019073:HT29 Cells; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors*; D016086:Lentivirus / Q000187:drug effects* / Q000235:genetics*; D016180:Lentivirus Infections / Q000821:virology; D046228:Microarray Analysis; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference / Q000187:drug effects*; D012367:RNA, Viral / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012371:Robotics; D014162:Transfection
chemical_list: D012367:RNA, Viral; D016547:Kinesins