Identification of skeletal muscle satellite cells by transfecting EGFP driven by skeletal alpha-actin promoter

In isolating skeletal muscle satellite cells, sometimes a problem is encoun tered in removing contaminating nonmyogenic cells. In the present study, we constructed a novel vector, pSKA-EGFP, which achieves the expression of en hanced green fluorescent protein (EGFP) exclusively in myogenic cells under the control of skeletal a-actin promoter when transfected to primary cultu red cells from skeletal muscle. Cells from rat skeletal muscle positive for EGFP after transfecting with pSKA-EGFP were all positive for desmin and no ne of the nonmyogenic cells expressed EGFP, indicating that the expression of EGFP is specific to myogenic cells. Among the cells positive for EGFP we re proliferating cells, presumably satellite cells. In addition, EGFP posit ive cells derived from horse skeletal muscle after transfecting pSKA-EGFP i n vitro formed multinuclear myotubes, indicating that myogenic expression o f EGFP driven by skeletal cc-actin was achieved also in the equine cells. T hese results indicated that pSKA-EGFP vector will be useful in identifying and following up the satellite cells in real time, and also permit us to is olate satellite cells in combination with fluorescence-activated cell sorti ng (FACS).