Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi.
Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets.
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
Johannes
J
Zuber
Katherine
K
McJunkin
Christof
C
Fellmann
Lukas E
LE
Dow
Meredith J
MJ
Taylor
Gregory J
GJ
Hannon
Scott W
SW
Lowe
D000818:Animals; D001709:Biotechnology / Q000379:methods; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D004195:Disease Models, Animal; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D007948:Leukemia, Monocytic, Acute / Q000628:therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D034622:RNA Interference / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
D005456:Fluorescent Dyes; D034741:RNA, Small Interfering; D015534:Trans-Activators; D013752:Tetracycline