Transfection is the process of introducing DNA into cells and expression of the gene contained in the DNA. The DNA itself could be a functional gene, part of a gene, or a gene with the regulatory and transcribed sequences intact. The ability to transfect mammalian cells is a powerful tool that can be used to study the function and control of mammalian genes. Transfection of DNA into eukaryotic cells can be done in several ways. One of the methods of introducing foreign DNA into mammalian cells is known as cationic liposome-mediated transfection. Preliminary studies involved the development of a cationic lipid-mediated transfection method using HeLa cells and a plasmid that codes for ~-galactosidase. The cationic lipid used was Transfectam. Transfectam is a commercially available, cationic lipopolyamine. Chitosan, a cationic polymer of glucosamine, was used as an alternative transfecting agent and as a binding agent for polyanions. The transfection efficiency of chitosan was compared to that of Transfectam. Chitosan was found to be comparable to Transfectam in this regard. Polyanions of different chemical structures were used in a chitosan binding study. These include aurin tricarboxylic acid, calf thymus DNA and methyl green DNA. The kinetics of the binding interactions suggest that ionic interactions predominated. Based upon these findings, studies were performed to determine the nature of chitosan interactions with the cell surface. Chitosan beads were prepared to determine the interaction of chitosan with the cell membrane of He La cells and for the isolation of membrane proteins. Membrane proteins which bound to chitosan beads could be effectively eluted with a-0-mannopyranoside but not sodium chloride. Furthermore, HeLa cells bound to the chitosan beads could be eluted with aD- mannopyranoside but not sodium chloride. The results suggested that membrane bound proteins interact with chitosan through carbohydrate moiety interactions which may facilitate the transfection process.
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-3319 |
| Date | 01 January 1997 |
| Creators | Venkatesh, Savitha |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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