A Northern blot demonstrating expression of recombinant human factor IX cDNA in C2/C12 mouse myoblasts.

Mouse myoblasts were plated in 6-well tissue culture plates at a density of 2.5 × 10⁵ cells per well. The myoblasts were grown for 2 days until they had reached confluence and then stimulated to differentiate into myotubes. After 6 days of differentiation, the myotubes were transduced with AAV particles of the different hF.IX constructs (MOI = 5 × 10⁴). Five days later, media and cellular RNA were harvested and analyzed by ELISA and Northern blot, respectively. Ten micrograms of RNA was examined for hF.IX (A) or b-actin (B) expression by Northern analysis and hybridization with the corresponding probes. The RNA samples loaded in each lane were lane 1, AAV-CMV-hF.IX3 (2.0 kb); lane 2, AAV-HSA-CMV-hF.IX2 (1.9 kb); lane 3, AAV-HSA-hF.IX3 (1.8 kb); lane 4, AAV-cTNT-hF.IX (3.0 kb); and lane 5, C2/C12 RNA only. The expected size for each expressed RNA is indicated in parentheses. (C) The levels of hF.IX transcript were normalized to the level of b-actin transcript, and the ratios are reported relative to the amount of hF.IX transcript expressed from the AAV-CMV-hF.IX-3 vector. The relative amount of hF.IX protein as assayed by ELISA are also reported.