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 encountered in removing contaminating nonmyogenic cells. In the present study, we constructed a novel vector, pSKA-EGFP, which achieves the expression of enhanced green fluorescent protein (EGFP) exclusively in myogenic cells under the control of skeletal alpha-actin promoter when transfected to primary cultured cells from skeletal muscle. Cells from rat skeletal muscle positive for EGFP after transfecting with pSKA-EGFP were all positive for desmin and none of the nonmyogenic cells expressed EGFP, indicating that the expression of EGFP is specific to myogenic cells. Among the cells positive for EGFP were proliferating cells, presumably satellite cells. In addition, EGFP positive cells derived from horse skeletal muscle after transfecting pSKA-EGFP in vitro formed multinuclear myotubes, indicating that myogenic expression of EGFP driven by skeletal alpha-actin was achieved also in the equine cells. These 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 isolate satellite cells in combination with fluorescence-activated cell sorting (FACS).
Yamanouchi
K
K
Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Soeta
C
C
Suzuki
S
S
Hasegawa
T
T
Naito
K
K
Tojo
H
H
D000199:Actins / Q000235:genetics*; D000818:Animals; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003893:Desmin / Q000032:analysis; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis; D014162:Transfection
D000199:Actins; D003893:Desmin; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins