Rapid purification of transfected porcine muscle cells.
A detailed methodology is described for fluorescence-activated cell sorting (FACS) of porcine muscle cells that have been transfected to express green fluorescent protein (GFP). Cells are liberated from porcine skeletal muscle and primary cultures are transfected with DNA encoding GFP. Primary cultures are subjected to immunocytochemistry using a primary muscle-specific monoclonal antibody followed by incubation with a phycoerythrin-conjugated second antibody. Transfected myoblasts are sorted from fibroblasts using forward angle light scatter and ninety degree light scatter, phycoerythrin fluorescence, and GFP fluorescence. These procedures allow for isolation of genetically- engineered porcine muscle cells more rapidly than traditional clonal selection procedures. Consequently, FACS provides porcine myoblast populations that retain the majority of their replicative capacity and are not contaminated with non-myogenic cells.
Department of Animal Science and Food Technology, Texas Tech University, Lubbock, TX, USA.
J R
JR
Blanton
J P
JP
Robinson
D E
DE
Gerrard
C A
CA
Bidwell
A L
AL
Grant
D000818:Animals; D000911:Antibodies, Monoclonal; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008164:Luminescent Proteins / Q000235:genetics*; D018482:Muscle, Skeletal / Q000166:cytology*; D010799:Phycoerythrin / Q000276:immunology; D013552:Swine
D000911:Antibodies, Monoclonal; D008164:Luminescent Proteins; D010799:Phycoerythrin; D049452:Green Fluorescent Proteins