Figure 4.

RecHTLV recombines loxHTLV in a genomic reporter in HeLa cells

(A) Schematic representation of the reporter cell line and expression constructs used for assessing RecHTLV activity in HeLa cells in a genomic context. The reporter construct consists of a puromycin cassette flanked by two loxHTLV sites that prevent the expression of mCherry driven by a spleen focus-forming virus (SFFV) promoter. Expression of mCherry is only possible upon recombination of the loxHTLV sites. In the bicistronic recombinase expression construct, the IRES (internal ribosomal entry site) allows simultaneous expression of the recombinase fused to a nuclear localization signal (NLS-Rec) and a GFP cassette driven by a CMV promoter. (B) Left: representative dot plots obtained by flow cytometry of the loxHTLV reporter cells transfected with Cre or RecHTLV expression constructs. On the right side, quantification of the recombination efficiency measured as the ratio of mCherry+ cells and the total transfected cells (GFP+). The bar shows the mean ± SD of three independent experiments (∗∗∗∗p < 0.0001; ns, not significant, unpaired two-sided t test). (C) PCR performed on genomic DNA from transfected loxHTLV HeLa reporter cells. Left: schematic representation of the PCR where primers F and R are located in the flanking regions outside loxHTLV. The expected amplification product is 1.2 kb for non-recombined and 560 bp for the recombined reporter. Right: agarose gel of the PCR products from genomic DNA extracted from different samples: lane 1, HeLa cells; lane 2–4, reporter HeLa cells transfected with the indicated construct; lane 5, water control. The upper band on the gel shows unrecombined reporter (two triangles) while the lower one shows recombination (one triangle). (D) Chromatogram of the Sanger sequencing from the recombined PCR band shown in (C) from the RecHTLV-treated sample.