Transduction and expression of rAAV-ET in skeletal muscle cells. (A) Structure of the rAAV-ET vector. ITR: AAV-2 inverted terminal repeats; tetO-CMV: tetracycline-inducible promoter including seven repeats of the tetracycline operator inserted upstream of the CMV minimal promoter; mEpo: murine erythropoietin cDNA; (A)n: SV4O bidirectional polyadenylation signal; rtTA: coding sequences for the tetracycline reverse transactivator; LTR: long terminal repeat of the MFG retrovirus construct. The BamHI fragment used as an Epo-specific probe is indicated. (B) Northern blot analysis of total RNAs extracted 7 days after exposure of human primary myotubes to rAAV-ET (2 × 10⁹ vector genomes/culture well). Cells were incubated in the presence (+) or in the absence (−) of doxycycline (1 μg/mL). (c) Control myotubes not exposed to rAAV-ET. Hybridization was performed with the combination of an Epo-specific and a rtTA-specific ³²P-labeled probe. Migration of 18S and 28S rRNAs is indicated. Amounts of RNAs loaded on each lane can be appreciated on the ethidium bromide staining of 28 S rRNA (28S).