Effects of androgens on rAAV transduction and attachment to human HuH7 cell line. (A) Human HuH7 hepatocarcinoma cells were cultured in serum-free hepatocyte culture media with (+DHT) or without (–DHT) 300 nM of DHT for 5 days prior to gene transfer. Cells from each treatment group (n = 5) were plated out at a density of 1 × 105 cells per well of 6-well plates and transduced at an MOI of 103 with rAAV-2 CAGG-FIX. Medium was conditioned for 24 hours and collected on day 4 after transduction and assayed for hFIX by ELISA. Error bars indicate SEM. (B) Fluorescence confocal micrographs of optical sections demonstrating attachment of Cy-3 (red fluorescence) labeled rAAV-2 CAGG-FIX to (i) untreated HuH7 cells, (ii) HuH7 cells treated with 300 nM/L DHT for 5 days, or (iii) CHO-K1 cells that lack HPG. The nuclei show a low level of green autofluorescence that was enhanced in these images simply to identify the whole cell. Original magnification, × 335 (i-ii) and × 178 (iii).
