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Rapid conditional knock-down–knock-in system for mammalian cells.

Nucleic Acids Res. 35:e17 (2007)
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Open Access Gold
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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.
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Publication type Article: Journal article
Document type Scientific Article
Keywords RIBOSOME BIOGENESIS; PROLIFERATION; NUCLEOLUS; MATURATION; COMPLEX
ISSN (print) / ISBN 0305-1048
e-ISSN 1362-4962
Quellenangaben Volume: 35, Issue: 3, Pages: , Article Number: e17 Supplement: ,
Publisher Oxford University Press
Reviewing status Peer reviewed