HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes

Analysis of RyR1 structure function in muscle cells is made difficult by th e low (<5%) transfection efficiencies of myoblasts or myotubes using calciu m phosphate or cationic lipid techniques. We inserted the full-length 15.3- kb RyR1 cDNA into a herpes simplex virus type 1 (HSV-1) amplicon vector, pH SVPrPUC between the ori/IE 4/5 promoter sequence and the HSV-1 DNA cleavage /packaging signal (pac). pHSVGN and pHSVGRyR1, two amplicons that expressed green fluorescent protein, were used for fluorescence-activated cell sorte r analysis of transduction efficiency. All amplicons were packaged into HSV -1 virus particles using a helper virus-free packaging system and yielded 1 0(6) transducing vector units/ml. HSVRyR1, HSVGRyR1, and HSVGN virions effi ciently transduced mouse myoblasts and myotubes, expressing the desired pro duct in 70-90% of the cells at multiplicity of infection 5. The transduced cells appeared healthy and RyR1 produced by this method was targeted proper ly and restored skeletal excitation-contraction coupling in dyspedic myotub es. The myotubes produced sufficient protein to allow single-channel analys es from as few as 10 100-mm dishes. In most cases this method could preclud e the need for permanent transfectants for the study of RyR1 structure func tion.