Fig. 1. Insulin-secreting cells are efficiently transduced using lentiviral vectors. (A) Lentiviral vectors that contained a self-inactivating deletion (SIN) in the LTR and the post-transcriptional regulatory element of the woodchuck hepatitis virus (Wpre) used the cytomegalovirus promoter (CMV) to drive the expression of either GFP or a Cx (32, 36 or 43) cDNA. (B) GFP fluorescence was analysed by FACS two days after transduction of RIN2A cells with a viral vector containing GFP coding sequence. The analysis revealed a marked increase in cell fluorescence, indicating efficient transduction of the GFP cDNA. The proportion of transduced cells (80%) was given by dividing the number of cells found in the region indicated by the double-headed arrow by the total cell number. Open area, uninfected controls; filled area, infected cells. (C) Monolayers of uninfected RIN cells and cells infected with lentiviral vectors encoding Cx32, Cx36 and Cx43 were immunostained using antibodies against these three Cxs. Whereas no Cx36 was detected in control cultures, this protein was abundantly expressed after transduction of the cognate cDNA. Similarly, Cx32 and Cx43 were detectable between most RIN cells 2 days after infection by the lentiviral vectors. Bar, 10 µm. (D) Western blots revealed Cx32, Cx36 and Cx43 in membrane extracts of transduced cells. By contrast, no specific band was detected in wild-type uninfected RIN2A cells. Membrane extracts of liver, Min6 cells and heart served as positive controls for Cx32, Cx36 and Cx43, respectively. Lanes containing membrane extracts of positive controls were loaded with 10 µg protein; other lanes were loaded with 1 µg protein. (E) Contrasting with the situation in wild-type RIN cells, which show no gap junctions (not shown), large gap junctional plaques were detected by freeze-fracture electron microscopy at membrane interfaces of RIN2A cells transduced with a lentiviral vector coding for Cx32, Cx36 or Cx43.