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 Citations (Scopus)

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

Fingerprint

Skeletal Myoblasts
Gene Transfer Techniques
Ryanodine Receptor Calcium Release Channel
Skeletal Muscle Fibers
Human Herpesvirus 1
Viruses
Muscle
Skeletal Muscle
Genes
Myoblasts
Virion
DNA Packaging
Helper Viruses
Packaging
Virus Assembly
Excitation Contraction Coupling
DNA Cleavage
Green Fluorescent Proteins
Muscle Cells
Transfection

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)

Cite this

Wang, Y., Fraefel, C., Protasi, F., Moore, R. A., Fessenden, J. D., Pessah, L. N., ... 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).

HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes. / Wang, Y.; Fraefel, C.; Protasi, F.; Moore, R. A.; Fessenden, J. D.; Pessah, L. N.; Difrancesco, A.; Breakefield, X.; Allen, P. D.

In: American Journal of Physiology - Cell Physiology, Vol. 278, No. 3 47-3, 2000.

Research output: Contribution to journalArticle

Wang, Y, Fraefel, C, Protasi, F, Moore, RA, Fessenden, JD, Pessah, LN, Difrancesco, A, Breakefield, X & Allen, PD 2000, 'HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes', American Journal of Physiology - Cell Physiology, vol. 278, no. 3 47-3.
Wang, Y. ; Fraefel, C. ; Protasi, F. ; Moore, R. A. ; Fessenden, J. D. ; Pessah, L. N. ; Difrancesco, A. ; Breakefield, X. ; Allen, P. D. / HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes. In: American Journal of Physiology - Cell Physiology. 2000 ; Vol. 278, No. 3 47-3.
@article{55b716c4f7524d1099ff39ccfa59d693,
title = "HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes",
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.",
keywords = "Excitation- contraction coupling, Helper virus-free herpes simplex virus type 1 packaging, Large complementary DNA, RyR1",
author = "Y. Wang and C. Fraefel and F. Protasi and Moore, {R. A.} and Fessenden, {J. D.} and Pessah, {L. N.} and A. Difrancesco and X. Breakefield and Allen, {P. D.}",
year = "2000",
language = "English (US)",
volume = "278",
journal = "American Journal of Physiology - Renal Fluid and Electrolyte Physiology",
issn = "1931-857X",
publisher = "American Physiological Society",
number = "3 47-3",

}

TY - JOUR

T1 - HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes

AU - Wang, Y.

AU - Fraefel, C.

AU - Protasi, F.

AU - Moore, R. A.

AU - Fessenden, J. D.

AU - Pessah, L. N.

AU - Difrancesco, A.

AU - Breakefield, X.

AU - Allen, P. D.

PY - 2000

Y1 - 2000

N2 - 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.

AB - 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.

KW - Excitation- contraction coupling

KW - Helper virus-free herpes simplex virus type 1 packaging

KW - Large complementary DNA

KW - RyR1

UR - http://www.scopus.com/inward/record.url?scp=0034057930&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034057930&partnerID=8YFLogxK

M3 - Article

C2 - 10712251

AN - SCOPUS:0034057930

VL - 278

JO - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

JF - American Journal of Physiology - Renal Fluid and Electrolyte Physiology

SN - 1931-857X

IS - 3 47-3

ER -