Figure 1

Transduction of locomotor muscles and the diaphragm with AAV9-d.n.FoxO. (A) Alignment of the dominant negative (d.n.) FoxO protein sequence, which includes amino acids 141–265 of mouse FoxO3a, with the corresponding mouse FoxO1 and FoxO4 amino acid sequences. FoxO1 shares ~85% amino acid sequence identity and FoxO4 75% sequence identity with the d.n.FoxO protein (shared amino acids denoted in green), all of which share >90% sequence conservation within this region. The 6 amino acid residues involved in DNA binding of the Forkhead Domain are highlighted in red and are denoted by hash marks (#) above the aligned sequences. (B and C) The AAV9 vectors driving expression of d.n.FoxO (or empty vector), which also contain an IRES driving the expression of GFP, were injected directly into the anterior hind limb compartment of mice to transduce the TA and EDL muscles, or injected directly into the intrathoracic cavity of mice to transduce the diaphragm. (B) Representative muscle cross-sections showing AAV9 transfection efficiency in the TA, EDL and diaphragm ~26 days post-injection as visualized via direct GFP fluorescence. (C) Confirmation that the d.n.FoxO protein was also expressed in muscles transduced with AAV9-d.n.FoxO was confirmed through western blot using an antibody against DsRed, which is fused to the d.n.FoxO protein.