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

Y. Wang, C. Fraefel, F. Protasi, R. A. Moore, J. D. Fessenden, L. N. Pessah, A. Difrancesco, X. Breakefield, P. D. Allen

Research output: Contribution to journalArticle

55 Scopus citations

Abstract

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 10 6 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume278
Issue number3 47-3
StatePublished - 2000
Externally publishedYes

Keywords

  • Excitation- contraction coupling
  • Helper virus-free herpes simplex virus type 1 packaging
  • Large complementary DNA
  • RyR1

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology
  • Physiology (medical)

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    Wang, Y., Fraefel, C., Protasi, F., Moore, R. A., Fessenden, J. D., Pessah, L. N., Difrancesco, A., Breakefield, X., & Allen, P. D. (2000). HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes. American Journal of Physiology - Cell Physiology, 278(3 47-3).