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...

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Bibliographic Details
Published in:Nucleic acids research 2007-02, Vol.35 (3), p.e17-e17
Main Authors: Hölzel, Michael, Rohrmoser, Michaela, Orban, Mathias, Hömig, Cornelia, Harasim, Thomas, Malamoussi, Anastassia, Gruber-Eber, Anita, Heissmeyer, Vigo, Bornkamm, Georg, Eick, Dirk
Format: Article
Language:English
Subjects:
Cell Line, Tumor
Gene Targeting - methods
Genes, Essential
Humans
Methods Online
MicroRNAs - metabolism
Mutation
Nuclear Proteins - antagonists & inhibitors
Nuclear Proteins - genetics
Nuclear Proteins - physiology
Proteins - antagonists & inhibitors
Proteins - genetics
Proteins - physiology
RNA Interference
RNA, Small Interfering - metabolism
Transfection
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Summary: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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkl1055