Cationic liposome-mediated transfection of mammary epithelial cells with AAV-based plasmid DNA
type: CreativeWork
description: Cationic liposome-mediated delivery of adeno-associated virus (AAV) based plasmid DNA has potential for efficient and safe delivery of DNA into a number of different cell types. The objective of this thesis was to develop techniques for transfecting adult somatic mammary epithelial cells with the aim of producing non-germline transgenic animals. Initially, chemical-mediated techniques were used to deliver plasmid DNA to mammary epithelial cells in vitro: this work was subsequently extended to in vivo studies. The research then focussed on cationic liposome-mediated delivery of AAV-based plasmid DNA to enhance transfection efficiency, and the molecular characteristics of AAV-based plasmid DNA as a vector for transfection and integration of DNA into the genome. Chemical-mediated procedures were used to deliver plasmid DNA to the mouse mammary epithelial cell line, HC11, and to primary bovine mammary cells in vitro. Four delivery vehicles were used; calcium phosphate co-precipitation, DOTAP, DMRIE-C and LipofectAMINE. The highest transfection efficiency resulted from using LipofectAMINE, although this efficiency was relatively low, with only 2.1% of cells found to express β-galactosidase. To establish the efficacy of chemical-mediated delivery of plasmid DNA in vivo and to investigate an intramammary route of delivery, lactating rats were transfected in vivo with plasmid DNA complexed with LipofectAMINE. These complexes were delivered by infusion through the mammary teat canal or by direct injection into the gland. The ensuing level of transfection was extremely low, with only a very small number of rat mammary epithelial cells found to express β-galactosidase. These results indicated that alternative transfection procedures were required. Inverted terminal repeats (ITRs) of AAV have previously been shown to enhance transfection efficiency, and these were included in a plasmid construct flanking a β-galactosidase reporter gene. LipofectAMINE was used to deliver this DN…
abstract: Cationic liposome-mediated delivery of adeno-associated virus (AAV) based plasmid DNA has potential for efficient and safe delivery of DNA into a number of different cell types. The objective of this thesis was to develop techniques for transfecting adult somatic mammary epithelial cells with the aim of producing non-germline transgenic animals. Initially, chemical-mediated techniques were used to deliver plasmid DNA to mammary epithelial cells in vitro: this work was subsequently extended to in vivo studies. The research then focussed on cationic liposome-mediated delivery of AAV-based plasmid DNA to enhance transfection efficiency, and the molecular characteristics of AAV-based plasmid DNA as a vector for transfection and integration of DNA into the genome. Chemical-mediated procedures were used to deliver plasmid DNA to the mouse mammary epithelial cell line, HC11, and to primary bovine mammary cells in vitro. Four delivery vehicles were used; calcium phosphate co-precipitation, DOTAP, DMRIE-C and LipofectAMINE. The highest transfection efficiency resulted from using LipofectAMINE, although this efficiency was relatively low, with only 2.1% of cells found to express β-galactosidase. To establish the efficacy of chemical-mediated delivery of plasmid DNA in vivo and to investigate an intramammary route of delivery, lactating rats were transfected in vivo with plasmid DNA complexed with LipofectAMINE. These complexes were delivered by infusion through the mammary teat canal or by direct injection into the gland. The ensuing level of transfection was extremely low, with only a very small number of rat mammary epithelial cells found to express β-galactosidase. These results indicated that alternative transfection procedures were required. Inverted terminal repeats (ITRs) of AAV have previously been shown to enhance transfection efficiency, and these were included in a plasmid construct flanking a β-galactosidase reporter gene. LipofectAMINE was used to deliver this DN…
author: Whyte, Brett
license: https://researchcommons.waikato.ac.nz/bitstreams/f014a980-a783-4daf-9025-8ca0bc07a5b7/download
date published: 2001
date modified: 2026-07-24T05:57:30Z
provider: University Waikato
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: The University of Waikato