A competitive cell growth assay for the detection of subtle effects of gene transduction on cell proliferation

J. J. M. Eekels, A. O. Pasternak, A. M. Schut, D. Geerts, R. E. Jeeninga, B. Berkhout*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

27 Citations (Scopus)

Abstract

RNA interference (RNAi) is a sequence-specific gene silencing mechanism with therapeutic potential against many human pathogens. To obtain a durable therapeutic effect, stable transduction of target cells with for instance a lentiviral vector that expresses a short hairpin (shRNA) inducer of the RNAi pathway is necessary. Apart from the intended therapeutic effect, this treatment can induce negative effects on cell proliferation via off-target effects. A careful evaluation of the transduced cells is required to develop a safe gene therapy approach. Stably transduced cells are usually selected by expression of the enhanced green fluorescent protein (GFP) marker. In this study we show that the mixed transduction culture, containing both transduced GFP(+) and untransduced GFP(-) cells, can simply be passaged to score the GFP(+)/GFP(-) ratio by longitudinal flow cytometric analysis as a measure of the negative impact of the RNAi treatment on the cellular proliferation rate. We show that this assay is sensitive, easy to use and internally controlled for assessing subtle effects on cell proliferation of lentiviral transduction and transgene expression.

Original languageEnglish
Pages (from-to)1058-1064
Number of pages7
JournalGene Therapy
Volume19
Issue number11
DOIs
Publication statusPublished - Nov-2012
Externally publishedYes

Keywords

  • lentiviral vector
  • transduction
  • cell growth
  • RNA interference
  • RNA
  • SIRNAS
  • HIV-1
  • INTERFERENCE
  • INHIBITION
  • INDUCTION
  • VECTORS
  • POTENT

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