Rapid conditional knock-down-knock-in system for mammalian cells.
RNA interference (RNAi) is a powerful tool to analyze gene function in mammalian cells. However, the interpretation of RNAi knock-down phenotypes can be hampered by off-target effects or compound phenotypes, as many proteins combine multiple functions within one molecule and coordinate the assembly of multimolecular complexes. Replacing the endogenous protein with ectopic wild-type or mutant forms can exclude off-target effects, preserve complexes and unravel specific roles of domains or modifications. Therefore, we developed a rapid-knock-down-knock-in system for mammalian cells. Stable polyclonal cell lines were generated within 2 weeks by simultaneous selection of two episomal vectors. Together these vectors mediated reconstitution and knock-down in a doxycycline-dependent manner to allow the analysis of essential genes. Depletion was achieved by an artificial miRNA-embedded siRNA targeting the untranslated region of the endogenous, but not the ectopic mRNA. To prove effectiveness, we tested 17 mutants of WDR12, a factor essential for ribosome biogenesis and cell proliferation. Loss-off function phenotypes were rescued by the wild-type and six mutant forms, but not by the remaining mutants. Thus, our system is suitable to exclude off-target effects and to functionally analyze mutants in cells depleted for the endogenous protein.
Hölzel
Michael
M
Institute of Clinical Molecular Biology and Tumour Genetics, GSF Research Center, Marchioninistrasse 25, 81377 Munich, Germany. hoelzel@gsf.de
Rohrmoser
Michaela
M
Orban
Mathias
M
Hömig
Cornelia
C
Harasim
Thomas
T
Malamoussi
Anastassia
A
Gruber-Eber
Anita
A
Heissmeyer
Vigo
V
Bornkamm
Georg
G
Eick
Dirk
D
D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D018390:Gene Targeting / Q000379:methods*; D020043:Genes, Essential; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D011506:Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins; D014162:Transfection
D018797:Cell Cycle Proteins; D035683:MicroRNAs; D009687:Nuclear Proteins; C484463:PES1 protein, human; D011506:Proteins; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C517873:WDR12 protein, human