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.

Baculovirus-mediated gene transfer is attenuated by sodium bicarbonate.

BACKGROUND: Baculovirus transduction of cultured mammalian cells is typically performed by incubating the cells with virus using culture medium (e.g. Dulbecco's modified Eagle's medium (DMEM)) as the surrounding solution. However, we previously uncovered that DMEM hinders the baculovirus-mediated gene transfer. METHODS: In this study, we systematically explored the influences of promoter and medium constituents on the transduction efficiency by using different recombinant viruses and surrounding solutions for transduction, followed by flow cytometric analyses. Whether the key medium component impeded baculovirus binding to the cells and subsequent virus entry was investigated by immunofluorescence/confocal microscopy and quantitative real-time polymerase chain reaction (Q-PCR). RESULTS: We demonstrated that the poorer transduction by using DMEM as the surrounding solution is independent of the promoter. Examination of the medium constituents group by group revealed that the balanced salt solution suppresses the baculovirus transduction. By omitting individual salt species in the balanced salt solution, we surprisingly uncovered that NaHCO(3), a common buffering agent, exerts the inhibitory effects in a concentration-dependent manner. Intriguingly, NaHCO(3) did not debilitate the baculovirus, nor did it inhibit virus binding to the cells. Instead, NaHCO(3) inhibited baculovirus transduction by reducing the intracellular virus number. CONCLUSIONS: To our best knowledge, this is the first report unraveling the significance of NaHCO(3) in gene transfer. Our finding suggests that baculovirus-mediated gene transfer can be readily enhanced by omitting NaHCO(3) from the medium during the transduction period.

Baculovirus transduction of rat articular chondrocytes: roles of cell cycle.

BACKGROUND: We have previously demonstrated highly efficient baculovirus transduction of primary rat articular chondrocytes, thus implicating the possible applications of baculovirus in gene-based cartilage tissue engineering. However, baculovirus-mediated gene expression in the chondrocytes is transient. METHODS: In this study, we attempted to prolong the expression by supertransduction, but uncovered that after long-term culture the chondrocytes became more refractory to baculovirus transduction. Therefore, the correlation between baculovirus-mediated enhanced green fluorescent protein (EGFP) expression and cell cycle was investigated by comparing the cycling chondrocytes and chondrocytes rich in quiescent cells, in terms of EGFP expression, virus uptake, cell cycle distribution, nuclear import and methylation of viral DNA. RESULTS: We demonstrated, for the first time, that baculovirus-mediated transduction of chondrocytes is correlated with the cell cycle. The chondrocytes predominantly in G2/M phase were approximately twice as efficient in EGFP expression as the cycling cells, while the cells in S and G1 phases expressed EGFP as efficiently as the cycling cells. Notably, the chondrocyte populations rich in quiescent cells resulted in efficient virus uptake, but less effective nuclear transport of baculoviral DNA and higher degree of methylation, and hence poorer transgene expression. CONCLUSIONS: These findings unravel the practical limitations when employing baculovirus in cartilage tissue engineering. The implications and possible solutions are discussed.

Variation of baculovirus-harbored transgene transcription among mesenchymal stem cell-derived progenitors leads to varied expression.

We have previously demonstrated that baculovirus can efficiently transduce human mesenchymal stem cells (MSCs) and MSCs-derived adipogenic, chondrogenic, and osteogenic progenitors without compromising the differentiation capacity. Remarkably, the transgene expression level and duration varied widely with the differentiation states at which the progenitors were transduced. However, whether the variation was a general phenomenon and what caused the variation were unclear. Here we demonstrated that transduction of the MSCs and MSC-derived progenitors using baculoviruses carrying egfp driven by CMV, EF-1alpha or CAG promoter resulted in a general trend of varied expression, that is, the chondrogenic progenitors allowed for the poorest expression while the adipogenic progenitors conferred the best expression. Quantification of the nuclear and cytoplasmic egfp gene copy numbers by quantitative real-time PCR revealed that the varied expression did not arise from the discrepancies in gene delivery efficiency nor was it due to the disparities in nuclear transport efficiency. In contrast, the transcription levels paralleled the overall expression levels. These data suggested that although the egfp genes could be efficiently delivered into the nuclei of chondrogenic progenitors, they were not transcribed as well as they were in the adipogenic progenitors. In conclusion, the rapidly altering cellular transcription machinery in the course of differentiation progression predominantly led to the varied expression levels.