Transplantation of Retroviral Producer Cells forIn VivoGene Transfer into Mouse Skeletal Muscle

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Abstract
We describe a new strategy for efficient in vivo gene transfer into skeletal muscle using retroviral vectors. Recombinant retroviral producer cells, previously treated with the cytostatic drug mitomycin C, were injected into regenerating muscle of adult nude, nude/mdx, and C57BL/10 mice. Using LacZ reporter gene activity, we detected efficient transduction in all mouse strains (Nude, mean 11%, range 4.2–21%; C57BL/10, mean 12%, range 3.4–20%; Nude mdx, mean 4.3%, range 2.1–7% at 4 weeks post-injection and 6.6%, range 1.3–12% at 12 weeks post-injection). Foreign gene expression was sustained at high levels for at least 3 months. This strategy allows muscle satellite cells to be transfected in vivo, forming a reservoir of the transgene for incorporation into new myofibers in subsequent rounds of degeneration and regeneration. Because of its efficiency and potentially broad application, this procedure represents a new strategy for in vivo genetic transfer in skeletal muscle and potentially in other tissues. Fassati et al. implanted retroviral producer cells, previously treated with mitomycin C to step cell division, in regenerating skeletal muscle of normal, nude, and nude/mdx mice. High transduction frequencies were observed in the tibialis anterior after 1 month using the reporter gene LacZ, and high levels of transgene expression were observed for at least 3 months. Three months after producer cell implantation, β-galactosidase (β-Gal)-positive, regenerating fibers were observed in Nude/mdx and after muscle dissociation, satellite cells were found to express the retrovirally transduced LacZ gene. Histochemical staining for β-Gal and the polymerase chain reaction (PCR) were used to exclude retroviral spread in spleens and testis of treated animals. The results indicated that, under appropriate conditions, implantation of producer cells is an efficient procedure to obtain in vivo retrovirus-mediated gene transfer in skeletal muscle.