Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

Brock University. Dept. of Biological Sciences. Thesis

Adenoviruses have been used as a model system for understanding gene expression, DNA replication, gene delivery and other molecular biological phenomenon. In this project, adenovirus was used as a model to study exogenous gene expression in mammalian cells. More specifically, several adenoviral components were identified to enhance gene expression together with components needed for viral DNA replication. The adenoviral elements that enhance gene expression were assembled in an expression vector (pEl). These include the viral inverted terminal repeats (ITRs), the El region, the major late promoter (MLP) and the tripartite leader sequence (TPL). The green florescence protein (GFP) was used as a reporter gene. Various aspects of gene expression were examined including DNA delivery and stability inside the cells as well as mRNA transcription and protein expression. First, the effect of DNA quality on its delivery, stability and expreSSIOn III mammalian cells was studied. Five different conditions of the major DNA contaminants were used in this investigation including ethidium bromide (EtBr) , cesium chloride (CsCl), EtBr/CsCl, endotoxins and ethanol. CsCl, EtBr/CsCl and endotoxins affected the delivery process while EtBr affected the expression process but not the delivery. The used EtOH had no significant effect on both. In addition, the effect of all the contaminants was reversible. Next, we looked at the factors that enhance mRNA transcription and translation levels. Three approaches were tested, the first was the co-transfection of pEl and a plasmid that contains adenoviral genes involved in replication (PE2: contains E2 and viral protease). The second was the establishment of a cell line expressing these adenoviral genes involved in replication and the third approach was the super-infection with the wild type adenovirus. The co-transfection did not show any significant increase in gene expression or vector stability. On the other hand, the construction of CHO-E2 cell lines yielded five cell lines but none of them showed expression of all the integrated adenoviral E2 genes or enhancement of stability. Adenoviral super-infection enhanced gene expression. CHO cells showed higher enhancement in intensity and time than human embryonic kidney (HEK) 293 cells. In addition, such enhancement was dependent on the multiplicity of infection (MOl). Finally, this study emphasizes the importance of DNA quality on gene expression. However, the use of adenoviral elements to enhance exogenous gene expression is successful only when the complementary viral proteins and sequences are present. Active expression of the adenoviral proteins does not depend on a few major elements, but depends on the combination of different elements that work in cis or trans to activate gene expression.