Highly efficient baculovirus-mediated gene transfer into rat chondrocytes.
To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (<1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25 degrees C), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes.
Ho
Yi-Chen
YC
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
Chen
Huang-Chi
HC
Wang
Kuei-Chun
KC
Hu
Yu-Chen
YC
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis*; D023822:Tissue Engineering / Q000379:methods*; D014161:Transduction, Genetic / Q000379:methods*
D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins