Figure 3.

Gene Therapy in Sgca null Mice

(A) Time course of development of centronucleation in gastrocnemius muscles of untreated Sgca null mice. CNF, centronucleation frequency. Each dot represents one mouse, and n = 4–6 per group. (B) Time course of development of Evans blue dye permeability in gastrocnemius muscles of untreated Sgca null mice. Each dot represents one mouse, and n = 6 per group. (C) Luciferase live imaging of representative animals in Sgca null mice. Image was taken 1 month after injection in gastrocnemius muscles of HBSS alone or SGCA + FST plasmids. (D) Western blot of same experiment described in (C). An antibody against the FLAG tag fused to the SGCA gene on the plasmid was used to detect expression of human SGCA. Protein extracted from C57BL/6 mice was used as the negative control for FLAG tag (lane 1). GAPDH was used as a loading control. (E) Western blot of follistatin delivery, from same experiment described in (C). Representative animals received the SGCA and FST plasmids, FST alone positive control (lane 2), or SGCA (negative control, lane 3). (F) ELISA of follistatin delivery, from same experiment described in (C). C57BL/6 and HBSS represent negative controls, while lane 3 animals were treated with the SGCA + FST plasmids. Data are mean ± SEM with n = 12 and *p < 0.05. (G) Evans blue dye analysis of 1- and 2-month studies of SGCA+FST. In both studies, muscles that received the SGCA and FST plasmids had significantly less penetration of Evans blue dye in muscle fibers, compared to HBSS-treated Sgca null mice. Data are mean ± SEM with n = 4–9 for the 1-month and n = 4–12 for the 2-month experiment. *p < 0.05. (H) Evans blue dye analysis of 1-month study in Sgca null mice injected in gastrocnemius muscles with plasmids encoding either SGCA, FST, or SGCA + FST, versus untreated mice. All plasmid-injected mice had a significant drop in EBD 1 month after injection. Data are mean ± SEM, with n = 4 and *p < 0.05.