Engineered CHO cell line with coxsackie and adenovirus gene enhance the susceptibility of adenovirus infection

• Main Author: Baba, Sayang
• Format: Thesis
• Language: English
• Published: 2013
• Subjects: QR Microbiology
• Online Access: http://eprints.utm.my/id/eprint/33781/5/SayangBabaMFBB2013.pdf; http://eprints.utm.my/id/eprint/33781/

Gene-based therapies promise the potential to target the explicit gene delivery and expression to target cell populations. Adenoviral vectors are presently being tested clinically as a new strategy for the treatment of cancer. However, an important determining factor for the successful entry of such adenoviruses into target cells is expression of the Coxsackie virus and Adenovirus Receptor (CAR) at the cell surface. This raises the possibility that those gene-based therapy face the greatest therapeutic challenge might be the least susceptible to infection with therapeutic adenoviruses. If effective strategies can be implemented to boost CAR expression and hence the presence of primary receptor at the cell surface, this could prove most useful to adenovirus-based gene transfer. Therefore, this study aimed to possibly boost the expression of CAR specifically on CAR-negative Chinese Hamster Ovary (CHO) cell lines and evaluate their biological function by examining their susceptibility to transduction and infection of Adenovirus type 5. Initially, CHO cell line were transfected with human CAR cDNA fragment tagged with red fluorescent protein (DsRed) and sequentially selected with G418 antibiotic to generate stable cell line containing CAR-DsRed. Several transfection reagents such as GeneJuice (Novagen), LipofectamineTM 2000 (Invitrogen) and Xtreme HP plasmid DNA (Roche) were used for transfection study using flow cytometry. In addition, the expression of CAR on CHO-CAR-DsRed cell was determined by inverted fluorescent microscope, qRT-PCR and western blotting and antibody-blocking assay to verify the role of CAR in CHO-CAR-DsRed cells. CHO-CAR-DsRed cells were further examined by infecting wild-type adenovirus type 5 (wt-Ad5) and transduction recombinant adenovirus type 5 tagged with enhanced green fluorescent protein (Ad5EGFP) and their susceptibility were observed by using cytopathic effect and Giemsa staining. Flow cytometry analysis, showed that transfection efficiency of Xtreme HP (39.25±1.00 MFI) was higher than LipofectamineTM 2000 (17.1±1.00 MFI) and GeneJuice (11.1±1.00 MFI). The stable CHO-CAR-DsRed cells were estimated at average of 68.9%±1.12 MFI by transduction of Ad5EGFP and supported by evidence of infectibility of wt-Ad5 from Giemsa staining. Moreover, blocking of CAR expression on CHO-CAR-DsRed showed negative susceptibility to Ad5EGFP. These findings suggested that the strategy could be implemented to augment CAR expression and enhance the presence of primary cell surface receptor, as this could ornament the cell’s susceptibility to adenovirus infection and beneficial for adenovirus-based gene therapy.