Journal Article

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

Michael Hölzel, Michaela Rohrmoser, Mathias Orban, Cornelia Hömig, Thomas Harasim, Anastassia Malamoussi, Anita Gruber-Eber, Vigo Heissmeyer, Georg Bornkamm and Dirk Eick

in Nucleic Acids Research

Volume 35, issue 3, pages e17-e17
Published in print February 2007 | ISSN: 0305-1048
Published online December 2006 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gkl1055
Rapid conditional knock-down–knock-in system for mammalian cells

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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.

Journal Article.  4165 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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