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 þ and untransduced GFP À cells, can simply be passaged to score the GFP þ /GFP À 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.

Selection of RNAi-based inhibitors for anti-HIV gene therapy

“…One could for instance determine the cellular doubling time by frequent cell counting. We recently developed a very user-friendly and ultra-sensitive assay that follows over time the ratio of shRNA-expressing GFP-positive cells vs untransduced GFP-negative cells in a co-culture assay [50] . This competitive cell growth or CCG assay has some clear advantages over other well-established cell proliferation assays: (1) After cell transduction, only a small aliquot of the culture is needed to launch the CCG assay, without any extra steps; (2) The CCG assay is internally controlled as it starts with a mixture of transduced and untransduced cells; and (3) Even minor effects on the cellular proliferation rate caused by shRNA expression can be detected.…”

Gene Therapy Strategies to Block HIV-1 Replication by RNA Interference

“…For instance, we tested the impact of lentivirus-transduced anti-HIV shRNAs cells and initially reported the absence of severe adverse effects [ 114 ]. Using the very sensitive cell competition concept as described above [ 113 ], we noticed a transient negative effect of one of the four shRNAs on T cell development (Fig. 9b ), which allowed us to reformulate the shRNA cocktail [ 115 ].…”

“…As mentioned, silencing of a cellular cofactor can affect cell growth. Although there are several possibilities to score cell growth over time, we realized the need for a simple assay to score subtle cell growth effects and developed the Competitive Cell Growth (CGG assay) [ 113 ]. This method is based on the difference in proliferation rate of transduced (GFP-positive) and untransduced cells in the same culture.…”

MAP3K11 is a tumor suppressor targeted by the oncomiR miR-125b in early B cells

“…To investigate their individual contribution to the oncogenic activity of miR-125b, we reconstituted three independent clones of miR-125b-transformed cells with cDNA expression constructs encoding the 11 target gene candidates and monitored green fluorescent protein (GFP) as an expression marker in a competitive growth assay over time (Figure 5a). 33 The expression constructs lacked a 3′-UTR and were therefore uncoupled from miR-125b-mediated regulation. We hypothesized that reconstitution of a target whose repression is involved in maintenance of the transformed state should counteract miR-125b function and reduce the percentage of GFP + cells.…”

“…53 The fitness of transduced cells compared with non-transduced controls in the same sample was analyzed based on a competitive growth assay. 33 Changes in fluorescent marker expression over time were converted into a growth defect, based on an estimated cell doubling time of 1 day.…”