Sustained baculovirus-mediated expression in myogenic cells.
BACKGROUND
Baculovirus has emerged as a promising gene delivery vector due to its low cytotoxicity and nonreplication nature in mammalian cells. However, baculovirus-mediated expression is transient and generally lasts less than 14 days, which could restrict its application in the treatment of diseases requiring stable transgene expression.


METHODS
We transduced myoblast cell lines C2C12, Sol 8 and primary myoblasts with a baculovirus expressing the enhanced green fluorescent protein (EGFP) under the control of cytomegalovirus immediate-early promoter and measured the transduction efficiency by flow cytometry. Myogenic differentiation was induced after transduction and the longevity of EGFP expression was monitored by fluorescence microscopy. The myogenic differentiation was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The persistence of the egfp DNA and transcripts was monitored by real-time PCR and quantitative real-time RT-PCR.


RESULTS
Baculovirus efficiently transduced C2C12, Sol 8 and the primary myoblasts. The transgene expression persisted for a prolonged period of time (at least 63 days) in the cells differentiating into myotubes, but was transient in HeLa cells (<7 days). The sustained expression paralleled the myogenic differentiation and stemmed from the intracellular persistence of egfp DNA and mRNA.


CONCLUSIONS
The transgene delivered by baculovirus persists in the myotubes and endows sustained expression, which is distinct from its rapid degradation and transient expression in other cell types. These findings justify the future use of baculovirus for muscle-based gene therapy.
Shen
Heng-Chun
HC
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
Yeh
Chia-Ni
CN
Chen
Guan-Yu
GY
Huang
Shiu-Feng
SF
Chen
Chi-Yuan
CY
Chiu
Yung-Chung
YC
Hu
Yu-Chen
YC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002454:Cell Differentiation; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D032446:Myoblasts / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes
D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA