Fig. 9
Regulatable expression of eGFP in primary HUVEC cells transduced with a SIN lentiviral vector. (A) Structure of SIN HIV vector constructs. The 1PcN and 1PiN cassettes were cloned into a SIN HIV-1 vector within the third deletion in the envelope region. The viral backbone contains a truncated gag/pol region and, vif and vpr open reading frames. The triangle in the U3 region of the 3′ LTR indicates the SIN deletion. (B) Efficiency of eGFP expression in HUVEC cells by FACS analysis. Cells (2 × 105 cells/well into six-well plates) were infected with 1PcN (panels 1 and 2) or 1PiN (panels 3 and 4) VSV-G pseudotyped SIN vectors using equal numbers of RT units (50,000 cpm) and in the presence of 20 µg/ml of DEAE-dextran. Four hours postinfection, cell monolayers were washed and fed with fresh media without (panels 1 and 3) or with (panels 2 and 4) 1 µg/ml of Tc. Sixteen days postinfection, cells were tested for expression of the reporter gene by FACS analysis. (C) Effects of delay in Tc treatment on eGFP induction and of removal of Tc on reversibility of eGFP induction. Mock-transduced cells (panel 1) and cells transduced with the 1PiN vector (panels 2–5) were maintained in culture for 16 days. Expression of eGFP in the absence (panel 2) or presence (panel 3) of Tc was analyzed by FACS analysis. At day 7, a portion of transduced HUVECs was harvested (panels 4 and 5). Cells that were maintained in the absence of Tc were fed with media containing the antibiotic (panel 4); withdrawal of Tc occurred to those that have been previously treated with Tc (panel 5). Incubation continued for another 9 days.