A facile lentiviral vector system for expression of doxycycline-inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha.
RNA interference (RNAi) is a powerful genetic tool for loss-of-function studies in mammalian cells and is also considered a potentially powerful therapeutic modality for the treatment of a variety of human diseases. During the past 3 years a number of systems for conditional RNAi have been developed that allow controlled expression of short hairpin RNA (shRNA) triggers of RNAi. The simplest strategy relies on tet-operable polymerase III-promoted shRNAs and co-expression of the tetracycline regulatory protein, TetR. In this study we have combined these features into a single lentiviral vector that upon delivery to target cells allows robust induction of shRNAs, even with low levels of doxycycline; importantly, we show minimal leakiness in the absence of inducer. We have exploited the regulatory properties of our system by targeting an essential cellular gene, the nuclear RNaseIII endonuclease Drosha. Drosha is the core catalytic component of the "microprocessor complex" and cleaves the primary microRNA (miRNA) transcripts into their pre-miRNA hairpin intermediates. We anticipate that our vector will facilitate functional studies of miRNA biogenesis.
Aagaard
Lars
L
Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Amarzguioui
Mohammed
M
Sun
Guihua
G
Santos
Luis C
LC
Ehsani
Ali
A
Prydz
Hans
H
Rossi
John J
JJ
D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D034741:RNA, Small Interfering / Q000235:genetics*; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
D035683:MicroRNAs; D012322:RNA Precursors; D034741:RNA, Small Interfering; D008156:Luciferases; C517918:DROSHA protein, human; D043244:Ribonuclease III; D004318:Doxycycline