Figure 3.

Activity of RecHTLV in human cells in transient reporter assay

(A) Schematic representation of the reporter and expression constructs used for assessing RecHTLV activity in HeLa cells. The reporter construct pCAGGS-lox-pA-lox-mCherry consists of 3 poly(A) (pA) sequences flanked by two loxHTLV sites which prevent the expression of mCherry driven by a CAG promoter. Expression of mCherry is only possible upon recombination of the loxHTLV sites. The IRES (internal ribosomal entry site) bicistronic expression construct allows simultaneous expression of the recombinase fused to a nuclear localization signal (NLS-Rec) and a GFP cassette driven by a CMV promoter. (B) Representative images showing the fluorescence-based recombination reporter assay. HeLa cells were co-transfected with the indicated recombinase and the reporter construct. Only upon the expression of RecHTLV we observed expression of mCherry. Scale bars, 200 μm. (C) Left: representative dot plots obtained by flow cytometry of HeLa cells transfected with the loxHTLV reporter construct and Cre or RecHTLV expression constructs. On the right side, quantification of the recombination efficiency measured as the ratio of mCherry+ cells and frequency of transfected cells (GFP+). The bar shows the mean ± SD of five independent experiments (∗∗∗p < 0.001, unpaired two-sided t test).