FIG 2.

AAV2 and Ad5 transduction in Ext1-deficient cells in culture. (a) Analysis of GFP transgene expression following AAV transduction of control CHO K1 or EXT1-deficient CHO pgsD-677 cells. Cells were transduced with an AAV2 vector encoding GFP (AAV-GFP) for 1 h in reduced serum-medium. GFP expression was analyzed in cell extracts by immunoblotting (IB) 48 h after transduction (left side). Total RNA was analyzed for GFP mRNA expression by quantitative real-time RT-PCR. GFP mRNA levels are normalized to GAPDH mRNA levels in the same samples (right side). (b) Analysis of GFP protein and RNA expression in primary hepatocyte cultures isolated from Ext1^f/f control or Ext1^HEP mutant mice 48 h following transduction with AAV-GFP as in panel a. GFP mRNA levels are normalized to mouse GAPDH mRNA levels in the same samples. (c) Transduction of wild-type CHO K1 cells and EXT1-deficient mutant CHO pgsD-677 cells with AdTL, an Ad5 vector carrying a GFP transgene expression cassette and a luciferase transgene expression cassette. The cells were transduced in serum-reduced medium or in serum-reduced medium supplemented with 8 μg/ml of FX. Forty-eight hours posttransduction, cell lysates were analyzed for luciferase transgene expression. (d) Primary cultured hepatocytes from Ext1^f/f control or Ext1^HEP mutant mice were transduced with AdTLY477A, an Ad5 vector in which CAR binding is ablated, carrying the same GFP and luciferase transgene expression cassettes as AdTL, in the presence or absence of FX. Forty-eight hours following transduction, the cells were analyzed for GFP (by immunofluorescence) and for luciferase transgene expression. RLU, relative light units. Values are means ± SD relative to the value for the control (wild-type CHO K1 cells or control Ext1^f/f hepatocytes). *, P < 0.05; **, P < 0.01.