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

Nucleic Acids Res. 2007;35(3):e17. doi: 10.1093/nar/gkl1055. Epub 2006 Dec 14.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle Proteins
  • Cell Line, Tumor
  • Gene Targeting / methods*
  • Genes, Essential
  • Humans
  • 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
  • RNA-Binding Proteins
  • Transfection


  • Cell Cycle Proteins
  • MicroRNAs
  • Nuclear Proteins
  • PES1 protein, human
  • Proteins
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • WDR12 protein, human