Plasmid transfection and retroviral transduction of porcine muscle cells for cell-mediated gene transfer

Cell-mediated gene transfer is a potential tool for studying muscle growth, but efficient genetic manipulation and implantation strategies have not be en developed for pigs. The objectives of the present study were to determin e methods for transient and stable incorporation of reporter genes into por cine muscle cells and to investigate their use for cell-mediated gene trans fer in pigs. Porcine myoblasts and fibroblasts were isolated from muscle of 2-wk-old male pigs. Myogenic cell lines were identified using muscle-speci fic monoclonal antibodies, myotube fusion assays, and the presence of muscl e-specific markers (MyoD and desmin). Four commercial cationic liposomes (l ipofectAMINE, lipofectin, cellFECTIN, and DMRIE-C) were tested at different DNA:lipid ratios for their ability to transfect myoblasts and fibroblasts transiently with a luciferase reporter plasmid. LipofectAMINE resulted in t he greatest (P < .01) transient luciferase activity for both cell types. El ectroporation of cells for transient transfection resulted in less lucifera se activity than cationic transfection. Stable transfections were conducted using a green fluorescence protein (GFP) reporter plasmid containing the n eomycin resistance gene. LipofectAMINE transfection resulted in stable GFP expression in 1:16,000 myoblasts and 1:33,000 fibroblasts. Stable electropo ration resulted in efficiencies that were significantly lower than establis hed with cationic liposomes. Porcine cells were transduced with GFP using v esicular stomatitis virus glycoprotein G pseudotyped retrovirus and resulte d in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. These r esults show that cationic liposomes are superior to electroporation for tra nsfection, but retroviral transduction produced stable reporter gene expres sion in > 80% of porcine muscle cells. Transduced GFP-positive cells were s eparated from GFP-negative cells by fluorescence-activated cell sorting and implanted into 2-wk-old male pigs. On d 4, implanted muscles were removed and subjected to immunodetection of GFP protein. Fibroblast implantation re sulted in limited GFP expression within muscle, whereas myoblast implantati on resulted in GFP within muscle fibers. This suggests that cell-mediated g ene transfer is possible in porcine muscle and may be useful as an approach for studying muscle growth in pigs.