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 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.
Wang
Y
Y
Department of Anesthesia, Brigham and Women's Hospital, Boston 02115, Massachusetts.
Fraefel
C
C
Protasi
F
F
Moore
R A
RA
Fessenden
J D
JD
Pessah
I N
IN
DiFrancesco
A
A
Breakefield
X
X
Allen
P D
PD
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000502:physiology*; D014162:Transfection / Q000379:methods*
D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002118:Calcium