Liposome Vector Core is a research core which will also perform service for the individual projects. We have in the core a collection of about 20 different cationic liposome formulations. Rabbit synoviocytes and mouse lung endothelial cells will be tested for the best formulation for transfection. The transfection protocol will also be optimized. These data shall help the Projects on the gene therapy of synovium (Evans) and the pulmonary endothelium (Lazo). For the research part, we will study the mechanism of heterogeneity in the transfectability of cells in a population. Highly and poorly transfectable cells win be isolated by cell sorting and their interactions with liposome/DNA complex will be studied and compared. Specifically the subcellular distribution of the complex will be examined by electron microscopy and EM autoradiography. The locations of DNA and lipids will also be followed by measuring the radiolabels in various subcellular fractions after the cells are homogenized. We will specifically study the interactions of liposome/DNA complex with the mouse endothelial cells with the goal of examining the reasons why these cells are relatively difficult to transfect. Throughout these studies a CAT reporter gene driven by the prokaryotic T7 promoter will be extensively used to distinguish the process of gene delivery from that of gene expression. We have shown that this plasmid can be delivered into mammalian cells together with purified T7 RNA polymerase. Thus, the expression of the reporter gene is independent of the eukaryotic transcription factors.
Showing the most recent 10 out of 210 publications
