Dystrophin Cytochemistry in Mdx Mouse Muscles Injected with Labeled Normal Myoblasts

Abstract

A new technique enables correlation of dystrophin expression with the location of donor versus host nuclei in the same sections of mdx mouse muscle injected with normal myoblasts. Myoblasts from C57BL/6J mice or from humans were labeled with 0.01% fluoro-gold (FG) in Dulbecco's Modified Eagles Medium (DMEM) for 16 h at 37°C before myoblast transfer. About 3 × 104 myoblasts were injected into the quadriceps muscles of mdx mice immunosuppressed with cyclosporine A (CsA). At 11, 21, or 25 days after myoblast transfer, injected muscles were dissected out and sectioned. These mouse sections were processed for dystrophin and then labeled with a fluorescent nucleus counterstain, 5 μg% Hoechst 33342 in phosphate-buffered saline (PBS), for 10 min at room temperature. Fluoro-gold labeling corresponding with Hoechst 33342 staining indicated survival of normal nuclei in dystrophic muscle. Dystrophin was found in the sarcolemma of myofibers containing FG-labeled nuclei but not of myofibers containing only Hoechst 33342-labeled nuclei. Control muscle samples showed neither FG labeling nor dystrophin. This study demonstrates that the donor human and mouse myoblasts survived and developed in host mouse muscles for at least 25 days after myoblast transfer, and that the localization of their normal nuclei correlates with dystrophin expression in muscle fibers of immunosuppressed mdx host mice.