McMillan, Andrew Kenneth; (1995) Liposome and polycation-transferrin-conjugate mediated delivery of DNA for the transient transfection of haemopoietic cell lines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The aim of this work was to develop a non toxic transfection protocol suitable for the transient transfection of haemopoietic cell lines. There is a need for such a system since current methods of transfection of non-adherent haemopoietic cells are problematic. The standard method of transfection is electroporation but, to be effective, it is necessary to kill over 80[percent] of the cells treated, so the small surviving population of cells may not be representative of the original population. I initially optimised the electroporation of various haemopoietic cell lines. I then used fluorescent dyes as a model system for macromolecular delivery and investigated various liposome methods for the delivery of DNA, including hybrid vesicles with reconstituted Sendai virus membrane proteins. I showed that fluorescent dyes could be associated with cells but that delivery to the cytosol was inadequate for the efficient transient expression of introduced DNA. I investigated reasons why adequate expression was not seen and adopted a number of strategies to try to correct this. An alternative means of transient transfection in haemopoietic cells with low toxicity is the use of transferrin conjugated to polycation as a DNA delivery system (Transferrinfection). These conjugates bind DNA by a polycation-DNA interaction and undergo receptor mediated endocytosis. In K562 and TF1 cells transferrinfection allows efficient expression of the reporter gene chloramphenicol-acetyl-transferase with virtually no cellular toxicity. Transfection is improved by the pre-incubation of the cells in an iron- chelator and the effect of the 3-Hydroxypyridin-4-one iron chelator Cp 94 and desferrioxamine was compared. An efficient non toxic transfection system would enable the study of the mechanisms involved in the proliferation and differentiation of haemopoietic cells. Polycation-transferrin conjugates are able to mediate transient expression of a reporter gene and therefore may be valuable in future studies.

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