Figure 1

Expression of EGFP, TK, TK/EGFP and E6mut in HeLa cells after transduction with Ad5-EGFP, Ad5-TK, Ad5-TK/EGFP and Ad5-E6mut. (A) Restriction map of pAd5CMV/TCS and of the 4 different inserts. (B) HeLa cells were seeded on 24 well plates (1.2 × 10⁵ cells/well) and transduced the next day with the different Ad vectors at a MOI of 1000. The TK/EGFP encoded fusion protein consisted of the entire TK protein at the N-terminus, a peptide linker SFKST and the complete EGFP protein at the C-terminus. Western-blotting analyses were carried out as described, using rabbit anti-TK antibody (obtained from William C. Summers, Yale University, New Haven, dilution 1/1300), mouse anti-EGFP antibody (Roche Diagnostics, dilution 1/1000), rabbit anti-Flag antibody (Sigma, dilution 1/2000) or mouse anti-HPV16 E6 protein antibody (1/500) [11]. (C) HeLa cells were seeded on 24 well plates and transduced as described above. Cells were fixed and treated for immunofluorescence microscopy as described [11] with anti-TK antibody (1/1300), with anti-Flag antibody (1/1000), or with anti-E6 antibody (1/1000) and with a goat anti-rabbit antibody coupled to Alexa 568 (Molecular Probes, dilution 1/1000) or goat anti-mouse antibody coupled to Alexa 488 (Molecular Probes, dilution 1/1000). The nuclei were stained with Hoechst 33342 for 5 min at room temperature. Cells were viewed using a Zeiss Axioplan microscope (D) Cells were seeded and transduced as described above. Forty-eight hours after infection, cells were incubated, or not, with ganciclovir (GCV) at 20 μg/mL. Four days later, surviving cells were analyzed using the MTT test (M2003, Aldrich-Sigma, St Quentin Fallavier, France) as described previously [12]. This test was performed in triplicates, error bars are standard deviations.