A fluorescent reporter gene as a marker for ventricular specification in ES-derived cardiac cells

We have established a CGR8 embryonic stem (ES) cell clone (MLC2ECFP) which expresses the enhanced cyan variant of Aequorea victoria green fluorescent protein (ECFP) under the transcriptional control of the ventricular myosin light chain 2 (MLC2v) promoter. Using epifluorescence imaging of vital embr yoid bodies (EB) and reverse transcription-polymerase chain reaction (RT-PC R), we found that the MLC2v promoter is sn itched on as early as day 7 and is accompanied by formation of cell clusters featuring a bright ECFP blue f luorescence. The fluorescent areas within the EBs were all beating on dag 8 , MLC2ECFP ES cells showed the same time course of cardiac differentiation as mock ES cells as assessed by RT-PCR of genes encoding cardiac-specific t ranscription factors and contractile proteins. The MLC2v promoter conferred ventricular specificity to ECFP expression within the EB as revealed by ML C2v co-staining of ECFP fluorescent cells, MLC2ECFP-derived cardiac cells s till undergo cell division on day 12 after isolation from EBs but withdraw from the cell cycle on day 16, This ES cell clone provides a powerful cell model to study the signalling roads of factors regulating cardiac cell prol iferation and terminal differentiation with a view to using them for experi mental cell therapy. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.