Mislick, Kimberly Ann (1996) The role of proteoglycans in the delivery of cationic-DNA complexes and enhanced delivery by folate receptor-mediated endocytosis. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-09182006-152253
Experiments were conducted to elucidate the mechanisms of polylysine-mediated transfection into mammalian cells in vitro. In the first chapter, the role of membrane-associated proteoglycans in transfection was investigated by testing transfection efficiency under a number of assay conditions. Cells were treated with sodium chlorate in order to desulfate glycosaminoglycan chains. Chlorate treatment inhibited expression of luciferase, the intracellular uptake of DNA, and binding of DNA to the cell surface. Expression, uptake, and binding of DNA was also inhibited by exogenous glycosaminoglycans and by glycosaminoglycan lyases. Similarly, each of the transfection steps was severely inhibited in CHO cell mutants, incapable of synthesizing proteoglycans. Transfection by certain cationic liposomes was also inhibited in the mutant cell line. The possible implications of these results for gene therapy of diseases affecting hematopoietic cells is described. In the second chapter, polylysine-mediated gene delivery via the folate receptor was developed. Gene delivery by folate receptor-mediated endocytosis was approximately 18 times higher than by nonspecific endocytosis of polylysine-DNA in the presence of chloroquine. A number of controls confirmed that the folate receptor was critical in the gene delivery mechanism. When chloroquine was removed from the media, transfection efficiency dropped approximately 30 fold, suggesting that gene delivery occurred via a lysosomal pathway. In the final chapter, transfection by folate receptor-mediated endocytosis was characterized by 2-photon laser scanning microscopy. KB cells were transfected with folate-polylysine-DNA complexes labeled with YOYO, a dimeric cyanine intercalator, and a single cell was examined over a two hour period. Although additional controls are required, preliminary evidence suggests that mitosis is not a strict requirement for delivery of DNA into the nucleus of cells.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||24 May 1996|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||18 Sep 2006|
|Last Modified:||26 Dec 2012 03:01|
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