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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Main Authors:	Hölzel, Michael, Rohrmoser, Michaela, Orban, Mathias, Hömig, Cornelia, Harasim, Thomas, Malamoussi, Anastassia, Gruber-Eber, Anita, Heissmeyer, Vigo, Bornkamm, Georg, Eick, Dirk
Format:	Online
Language:	English
Published:	Oxford University Press 2007
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1807947/

id	pubmed-1807947
recordtype	oai_dc
spelling	pubmed-18079472007-03-02 Rapid conditional knock-down–knock-in system for mammalian cells Hölzel, Michael Rohrmoser, Michaela Orban, Mathias Hömig, Cornelia Harasim, Thomas Malamoussi, Anastassia Gruber-Eber, Anita Heissmeyer, Vigo Bornkamm, Georg Eick, Dirk Methods Online 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. Oxford University Press 2007-02 2006-12-14 /pmc/articles/PMC1807947/ /pubmed/17169998 http://dx.doi.org/10.1093/nar/gkl1055 Text en © 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Hölzel, Michael Rohrmoser, Michaela Orban, Mathias Hömig, Cornelia Harasim, Thomas Malamoussi, Anastassia Gruber-Eber, Anita Heissmeyer, Vigo Bornkamm, Georg Eick, Dirk
spellingShingle	Hölzel, Michael Rohrmoser, Michaela Orban, Mathias Hömig, Cornelia Harasim, Thomas Malamoussi, Anastassia Gruber-Eber, Anita Heissmeyer, Vigo Bornkamm, Georg Eick, Dirk Rapid conditional knock-down–knock-in system for mammalian cells
author_facet	Hölzel, Michael Rohrmoser, Michaela Orban, Mathias Hömig, Cornelia Harasim, Thomas Malamoussi, Anastassia Gruber-Eber, Anita Heissmeyer, Vigo Bornkamm, Georg Eick, Dirk
author_sort	Hölzel, Michael
title	Rapid conditional knock-down–knock-in system for mammalian cells
title_short	Rapid conditional knock-down–knock-in system for mammalian cells
title_full	Rapid conditional knock-down–knock-in system for mammalian cells
title_fullStr	Rapid conditional knock-down–knock-in system for mammalian cells
title_full_unstemmed	Rapid conditional knock-down–knock-in system for mammalian cells
title_sort	rapid conditional knock-down–knock-in system for mammalian cells
description	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.
publisher	Oxford University Press
publishDate	2007
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1807947/
_version_	1611394657204305920

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

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