Co-detection of GFP and Dystrophin in Skeletal Muscle Tissue Sections

Abstract

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration that results from the absence of the cytoskeletal protein dystrophin. DMD cell-based therapy exploits the ability of various cell populations (muscle- and non-muscle-derived) to incorporate into diseased muscle and produce dystrophin. A common experimental approach involves the delivery of labeled donor cells and evaluation of their engraftment potential by quantifying expression of donor proteins in the muscles of recipient mice. Green fluorescent protein (GFP) is a unique protein with several positive features that make it an exceptional cell-tracking marker. GFP is easily detected, is genetically transmitted, diffuses rapidly in the cytoplasm of muscle fibers, and clearly delineates the morphology of the cell in which it is expressed. Most studies of DMD cell-based therapy used only GFP expression to measure donor cell engraftment into muscle, while some studies evaluated expression of GFP and dystrophin not in the same but in consecutive muscle sections (1,2). Here, we report a method for simulta-neous tracking of GFP fluorescence and dystrophin expression in the same muscle section.GFP-labeled donor cells were isolated from the skeletal muscle of C57BL/6-Tg(ACTB-EGFP)1Osb/J mice as previously described (3). Recipient mice, C57Bl6Ros-5cv (mdx