title: Cationic liposome-mediated transfection of mammary epithelial cells with AAV-based plasmid DNA

authors: Whyte, Brett

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 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.

text: 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.

year: 2001

title: Electron and proton heating by solar wind turbulence

authors: Breech, Ben

subjects: solar wind turbulence

abstract: Previous formulations of heating and transport associated with strong magnetohydrodynamic (MHD) turbulence are generalized to incorporate separate internal energy equations for electrons and protons. Electron heat conduction is included. Energy is supplied by turbulent heating that affects both electrons and protons and is exchanged between them via collisions. Comparison to available Ulysses data shows that a reasonable accounting for the data is provided when (1) the energy exchange timescale is very long and (2) the deposition of heat due to turbulence is divided, with 60% going to proton heating and 40% into electron heating. Heat conduction, determined here by an empirical fit, plays a major role in describing the electron data.

text: Electron and proton heating by solar wind turbulence Previous formulations of heating and transport associated with strong magnetohydrodynamic (MHD) turbulence are generalized to incorporate separate internal energy equations for electrons and protons. Electron heat conduction is included. Energy is supplied by turbulent heating that affects both electrons and protons and is exchanged between them via collisions. Comparison to available Ulysses data shows that a reasonable accounting for the data is provided when (1) the energy exchange timescale is very long and (2) the deposition of heat due to turbulence is divided, with 60% going to proton heating and 40% into electron heating. Heat conduction, determined here by an empirical fit, plays a major role in describing the electron data.

year: 2009

title: Tukua Kia Rere! Maori Adult Students Succeeding at University

authors: Williams, Daphne Christina (Tina)

subjects: Maori students in higher education

abstract: A considerable amount of research has focussed on Māori underachievement in mainstream education. There is, however, much less research that celebrates Māori academic success. Even less has been written about the experiences of adult Māori students who return to study long after leaving school and go on to become academically successful in higher education. This study contributes to the growing body of knowledge regarding Māori academic success at university. It sought to explore the experiences of sixteen adult Māori students who entered university via special admission and went on to attain undergraduate degrees. The study examined the challenges that these individual’s experienced on their way to becoming academically successful and identified the main factors that helped them to achieve educational success. An overarching Kaupapa Māori theoretical framework was employed in this study. A qualitative research methodology was implemented and Kaupapa Māori research principles informed both the research processes and the interpretation of research outcomes. The findings suggest four major factors contributed to the success of the participants in this study: a strong determination to succeed (whāia te iti kahurangi), the extended family (whānau), strong social support networks with peers and faculty (whakawhanaungatanga) and Te Ao Māori (the Māori World). However, some of these same factors were also found to hinder academic success in the university setting. The Manu Tukutuku (kite) model was developed directly from the research findings. In addition to providing a theoretical frame for understanding the experiences of Māori special admission students at university, this model also provides a useful ‘toolkit’ for those wanting to support Māori students within the university context. The implications of this study suggest that New Zealand Universities can better support Māori special admission students by being more inclusive of their cultural capital, by eliminating racism and discrimination in the university environment and by fostering better relationships between staff and students. Creating opportunities for Māori students to develop social networks at University was also identified as important.

text: Tukua Kia Rere! Maori Adult Students Succeeding at University A considerable amount of research has focussed on Māori underachievement in mainstream education. There is, however, much less research that celebrates Māori academic success. Even less has been written about the experiences of adult Māori students who return to study long after leaving school and go on to become academically successful in higher education. This study contributes to the growing body of knowledge regarding Māori academic success at university. It sought to explore the experiences of sixteen adult Māori students who entered university via special admission and went on to attain undergraduate degrees. The study examined the challenges that these individual’s experienced on their way to becoming academically successful and identified the main factors that helped them to achieve educational success. An overarching Kaupapa Māori theoretical framework was employed in this study. A qualitative research methodology was implemented and Kaupapa Māori research principles informed both the research processes and the interpretation of research outcomes. The findings suggest four major factors contributed to the success of the participants in this study: a strong determination to succeed (whāia te iti kahurangi), the extended family (whānau), strong social support networks with peers and faculty (whakawhanaungatanga) and Te Ao Māori (the Māori World). However, some of these same factors were also found to hinder academic success in the university setting. The Manu Tukutuku (kite) model was developed directly from the research findings. In addition to providing a theoretical frame for understanding the experiences of Māori special admission students at university, this model also provides a useful ‘toolkit’ for those wanting to support Māori students within the university context. The implications of this study suggest that New Zealand Universities can better support Māori special admission students by being more inclusive of their cultural capital, by eliminating racism and discrimination in the university environment and by fostering better relationships between staff and students. Creating opportunities for Māori students to develop social networks at University was also identified as important.

year: 2010