Improving gene therapy

• Main Author: Wang, Eliza Lisha
• Other Authors: Klaus Erik Karjalainen
• Format: Final Year Project
• Language: English
• Published: 2009
• Subjects: DRNTU::Engineering::Chemical engineering::Biotechnology
• Online Access: http://hdl.handle.net/10356/16911
• Institution: Nanyang Technological University

Retroviral mediated gene therapy has progressed along with advances in biological science. Existing PT67 and Amphotropic-Phoenix retroviral packaging cell lines can be made more efficient to produce higher retroviral titers, giving rise to more efficient gene transfer. Here, an IRES-mcherry gene was cloned into two env-expressing plasmid vectors - pAmpho and p10A1. Under the control of the cytomegalovirus (CMV) promoter and the internal ribosomal entry site (IRES), the expression of the envelope protein encoded by the env-expressing vectors would be indicated by the presence of red fluorescence from mcherry. Packaging cells expressing high levels of mcherry could then be selected to generate efficient packaging cell lines. However, transfection of the packaging cell lines with the recombinant envelope plasmids showed that the transfected cells either did not express mcherry, or, if they did, mcherry was expressed at a very low level. Retroviruses produced from these packaging lines transfected with the recombinant envelope plasmids were used to infect wild-type Jurkat T-cells. FACS analysis of these Jurkat T-cells indicated that the packaging lines did produce retroviral particles expressing the env protein. Although the yield of these retroviral particles did not increase with co-transfection of these packaging lines with the constructed envelope plasmids, the expressed env protein enhanced the efficiency of infection.