Cationic liposome-mediated transfection of mammary epithelial cells with AAV-based plasmid DNA
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 DNA to HC11 cells, however, the efficiency of transient transfection was only increased by a modest 1.4-fold. To evaluate the effect of AAV ITRs on plasmid DNA integration, a DNA construct containing a neomycin resistance gene flanked by ITRs was transfected into HC11 cells. Additionally, this construct was co-transfected with an AAV Rep protein expression construct, which is required for site-specific integration in human cells. Neither the inclusion of ITRs, or expression of the rep gene affected the frequency of integration of the neomycin gene. In humans a specific advantage of using AAV is site-specific integration of the virus into a locus on human chromosome 19, known as AAVS1. Southern blot analyses were used to ascertain whether a similar site is present in mouse and other species. No clear cut evidence was found for such a site. Further Southern blot analysis of clonal lines derived from mouse HC11 cells that were co-transfected with a neomycin resistance gene, and an AAV rep gene construct, suggested that the site of integration was random. A novel PCR technique was utilised to amplify the site of integration. This enabled sequence data to be obtained for two HC11 clones. The site of integration for one of the clones was identified within exon 16 of Mus musculus DNA coding for the DNA dependent protein kinase catalytic subunit. The site of integration for the second clone was an unknown region of the mouse genome. An interesting characteristic of both clones was the presence of mouse B1 repeat sequences in close proximity to the sites of integration. These studies demonstrate that AAV-based plasmid DNA can enhance the transfection efficiency in mouse mammary epithelial cells, and they contribute toward the overall knowledge of AAV-based plasmid DNA integration into the mouse genome.
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