Nucleic Acids Research Advance Access published online on December 14, 2006
Nucleic Acids Research, doi:10.1093/nar/gkl1055
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Methods Online |
Rapid conditional knock-down–knock-in system for mammalian cells
Institute of Clinical Molecular Biology and Tumour Genetics, GSF Research Center Marchioninistrasse 25, 81377 Munich, Germany 1 Institute of Molecular Immunology, GSF Research Center Marchioninistrasse 25, 81377 Munich, Germany
*To whom correspondence should be addressed. Tel: +49 89 709 9512; Fax: +49 89 709 9500; Email: eick{at}gsf.de
Received August 26, 2006. Revised November 7, 2006. Accepted November 17, 2006.
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.
*Correspondence may also be addressed to Michael Hölzel. Tel: +49 89 709 9512; Fax: +49 89 709 9500; Email: hoelzel{at}gsf.de
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