Highly efficient baculovirus-mediated gene transfer into rat chondrocytes

• Published in: Biotechnology and bioengineering Vol. 88; no. 5; pp. 643 - 651
• Main Authors: Ho, Yi-Chen, Chen, Huang-Chi, Wang, Kuei-Chun, Hu, Yu-Chen
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 05.12.2004; Wiley; Wiley Subscription Services, Inc
• Subjects: Animals; Baculoviridae - genetics; Baculovirus; Biological and medical sciences; Biotechnology; Cartilage (articular); Cell culture; Cell proliferation; Cells, Cultured; Chondrocytes; Chondrocytes - physiology; Chondrogenesis; Collagen (type II); Deoxyribonucleic acid; Differentiation; DNA; Fundamental and applied biological sciences. Psychology; gene delivery; Gene Targeting - methods; Gene transfer; Gene Transfer Techniques; Genes; Genetic Vectors; Glycosaminoglycans; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; Growth rate; mammalian cell; Polymerase chain reaction; Q1; Q2; rat chondrocytes; Rats; Rats, Wistar; Recombinant Proteins - biosynthesis; Rodents; Temperature effects; Tissue engineering; Tissue Engineering - methods; Transduction, Genetic - methods; mammalian cell; Chondrocyte; Rat; Rodentia; Genetic transfer; rat chondrocytes; Virus; gene delivery; Vertebrata; baculovirus; Mammalia; Baculoviridae; Gene
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20239

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°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. © 2004 Wiley Periodicals, Inc.