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1 Department of Anesthesia, Brigham and Women's Hospital, Boston 02115; 4 Molecular Neurogenetics Unit, Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02129; 2 University Institution for Virology, 8057 Zurich, Switzerland; and 3 Department of Molecular Biosciences, University of California, Davis, School of Veterinary Medicine, Davis, California 95616
Analysis of RyR1 structure function in muscle cells is made difficult by the low (<5%) transfection efficiencies of myoblasts or myotubes using calcium 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, pHSVPrPUC 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 sorter analysis of transduction efficiency. All amplicons were packaged into HSV-1 virus particles using a helper virus-free packaging system and yielded 106 transducing vector units/ml. HSVRyR1, HSVGRyR1, and HSVGN virions efficiently transduced mouse myoblasts and myotubes, expressing the desired product in 70-90% of the cells at multiplicity of infection 5. The transduced cells appeared healthy and RyR1 produced by this method was targeted properly and restored skeletal excitation-contraction coupling in dyspedic myotubes. The myotubes produced sufficient protein to allow single-channel analyses from as few as 10 100-mm dishes. In most cases this method could preclude the need for permanent transfectants for the study of RyR1 structure function.
helper virus-free herpes simplex virus type 1 packaging; RyR1; excitation-contraction coupling; large complementary DNA
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