Improvement of Gene Transduction Efficacy by Incorporating Insulator into VP16-GAL4-WPRE Integrated Systemic Amplifier (VISA) System

• Main Authors: Chien-Fu Chen, 陳建甫
• Other Authors: Chia-Ling Hsieh
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/26571165170066327247

碩士 === 中國醫藥大學 === 癌症生物學研究所碩士班 === 99 === Recently, a VISA vector has been developed to enhance the transcriptional activity of therapeutic gene without impairing tumor-targeting efficacy. Herein, we further improve the effectiveness of gene expression of VISA vector by incorporating insulator elements between two adjacent promoters to minimize the promoter interference. To select the optimal length of the cHS4 insulator, we tested various survivin-VISA plasmids by inserting 1.2 kb full length, 250 bp core element, and two tandem repeats of core element of cHS4 insulator between survivin promoter and G5E4T promoter in forward (+) and reverse (-) orientation. The transgene expression among constructs were determined and compared in survivin-expressing CL1-5 human lung cancer cell line and a survivin-null normal epithelial 184A1 cell line. One copy of core element either within the proximal 250 bp or full length of cHS4 insulator displays no significant increased luciferase activity by survivin-VISA vector in the forward orientation. However, around two-fold higher reporter expression was observed in the two tandem repeats of core element than survivin-VISA vector. In contrast, reverse orientation of insulator fragment in the survivin-VISA vector enhanced luciferase activity about 2-3 folds regardless of the region of core element in CL1-5 cells but remained no effect on 184A1 cells, this result also shown in oral cancer. We then determine cell cytotoxicity by construct trail into survivin-VISA vector. Insulated-survivin-VISA-trail and survivin-VISA-trail were determined the in vitro cell killing effect in both lung and oral cancer, insulated-survivin-VISA-trail was shown increased in vitro cell killing effect compared to survivin-VISA-trail. Combine insulated-survivin-VISA trail and radiotherapy could also sensitize CL1-5 and SAS oral cancer cell. Our data presents for the first time that insulator can be incorporated into dual-promoter based vectors to enhance transgene production from each expression cassette in the same construct. We are currently evaluating this newly developed VISA system into targeted gene therapy for its therapeutic potential.