title: A competitive cell growth assay for the detection of subtle effects of gene transduction on cell proliferation.
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.
authors: Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands.
authors: J J M
authors: JJ
authors: Eekels
authors: A O
authors: AO
authors: Pasternak
authors: A M
authors: AM
authors: Schut
authors: D
authors: D
authors: Geerts
authors: R E
authors: RE
authors: Jeeninga
authors: B
authors: B
authors: Berkhout
mesh_terms: D002460:Cell Line; D049109:Cell Proliferation; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D034622:RNA Interference; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D049452:Green Fluorescent Proteins