RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells

Ralf Kittler, Laurence Pelletier, Chunling Ma, Ina Poser, Steffi Fischer, Anthony A. Hyman, Frank Buchholz

Research output: Contribution to journalArticle

76 Citations (Scopus)

Abstract

RNA interference (RNAi) is a widely used method for analysis of gene function in tissue culture cells. However, to date there has been no reliable method for testing the specificity of any particular RNAi experiment. The ideal experiment is to rescue the phenotype by expression of the target gene in a form refractory to RNAi. The transgene should be expressed at physiological levels and with its different splice variants. Here, we demonstrate that expression of murine bacterial artificial chromosomes in human cells provides a reliable method to create RNAi-resistant transgenes. This strategy should be applicable to all eukaryotes and should therefore be a standard technology for confirming the specificity of RNAi. We show that this technique can be extended to allow the creation of tagged transgenes, expressed at physiological levels, for the further study of gene function.

Original languageEnglish (US)
Pages (from-to)2396-2401
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number7
DOIs
StatePublished - Feb 15 2005

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Bacterial Artificial Chromosomes
Gene Transfer Techniques
RNA Interference
Cell Culture Techniques
Transgenes
Eukaryota
Genes
Technology
Phenotype
Gene Expression

Keywords

  • Gene knockdown
  • Interferon response
  • Loss-of-function experiment
  • Off-target effects

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells. / Kittler, Ralf; Pelletier, Laurence; Ma, Chunling; Poser, Ina; Fischer, Steffi; Hyman, Anthony A.; Buchholz, Frank.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 7, 15.02.2005, p. 2396-2401.

Research output: Contribution to journalArticle

Kittler, Ralf ; Pelletier, Laurence ; Ma, Chunling ; Poser, Ina ; Fischer, Steffi ; Hyman, Anthony A. ; Buchholz, Frank. / RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells. In: Proceedings of the National Academy of Sciences of the United States of America. 2005 ; Vol. 102, No. 7. pp. 2396-2401.
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