Fig. 2

Stable and bicistronic expression of Tomato and GFP fluorescent proteins in forelimb muscles following chick limb somite electroporation. Forelimb somites of E2.5/HH15 chick embryos were electroporated with the pT2AL-CMV/βactin-Tomato-T2A-GFP stable vector containing the Tomato-T2A-GFP cassette under the control of a general promoter. Six days after electroporation, at E8.5, forelimbs were collected for wholemount visualisation (a-c), immunostaining on transverse (d–i) or longitudinal (j–r) limb sections. Both Tomato and GFP fluorescent proteins were expressed in forelimb muscles, visualised in whole mount embryos (a–c). The Tomato and GFP expression was visualised in all limb muscles on transverse limb sections (d–f). Higher magnifications of muscle transverse sections showed that GFP+ nuclei were associated with Tomato in membrane (g–i, arrowheads). However, Tomato was not always associated with GFP due to the multinucleated statute of muscle fibres and membrane fluidity (g–i, arrows). Longitudinal muscle sections showed electroporated muscle fibres displaying Tomato fluorescence in sarcolemma with only a subset of GFP+ myonuclei (j–l arrowheads). GFP- myonuclei are arrowed (j–l arrows). m–r Electroporated muscle cells co-expressing both Tomato at the membrane and GFP in nuclei were observed in MF20+ muscle fibres (m–o, arrowheads) and in Pax7+ progenitors (p–r, arrows)