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Using novel approaches to understand development: gene targeting, protein labelling and the development of the accessory glands in Drosophila melanogaster

Kearney, Kate

Description

Three very different approaches to understanding developmental biology were taken in this project. The first was an attempt to investigate the subcellular localisation of the Drosophila RhoGEF Pebble (Pbl) in live cells in real time, with an aim to further elucidate the role of Pbl in EMT and mesoderm migration in the embryo. This method, tetracysteine labelling, makes use of a 6 aa tag and a fluorescent, cell permeable, biarsenical ligand. Pioneered in Roger Tsien's laboratory, it utilises the...[Show more]

dc.contributor.authorKearney, Kate
dc.date.accessioned2018-11-22T00:05:36Z
dc.date.available2018-11-22T00:05:36Z
dc.date.copyright2009
dc.identifier.otherb2519515
dc.identifier.urihttp://hdl.handle.net/1885/150398
dc.description.abstractThree very different approaches to understanding developmental biology were taken in this project. The first was an attempt to investigate the subcellular localisation of the Drosophila RhoGEF Pebble (Pbl) in live cells in real time, with an aim to further elucidate the role of Pbl in EMT and mesoderm migration in the embryo. This method, tetracysteine labelling, makes use of a 6 aa tag and a fluorescent, cell permeable, biarsenical ligand. Pioneered in Roger Tsien's laboratory, it utilises the reversible covalent bonds that form between closely spaced pairs of thiols in the tag and arsenical compounds. However, it was unsuccessful in allowing precise localisation of the Pbl protein either in vitro in Drosophila S2 cell culture or in vivo in wholemount embryos. While the approach has had success in the mammalian system, particularly mammalian cell culture, it would appear as though wholemount Drosophila embryos are not amenable to this technology. Secondly, I attempted to delete the PH domain of Pbl using ends out targeted homologous recombination, with an aim to investigate the role of the PH domain in EMT and mesoderm migration in the embryo. The first targeting attempt, to introduce a selectable marker, was successful, with a targeting efficiency of 1 in 160,000. However, the insertion of the PH deletion proved unsuccessful, perhaps due to a small recombination loophole introduced in the first targeting event. Thirdly and finally the development of the binucleate accessory glands was investigated and found to result from a failure of cytokinesis following mitoses that took place between 60 hours and 74 hours after puparium formation. In order to develop tools for the genetic manipulation of this tissue, the expression pattern of two GAL4 drivers, btlGAL4 and prdmf9GAL4, was investigated. prdmf9GAL4 was found to be expressed highly and consistently throughout the binucleate cell formation period and into the adult gland. Whereas, btlGAL4 was more highly expressed in the associated tracheal tissue of the gland during accessory gland development than in the gland itself. Expression of GAL4 by both drivers induced the formation of mononucleate accessory gland cells at different developmental stages, although the reason for this was not elucidated. This mononucleate cell type persisted in some cells of the adult gland in the case of prdmf9GAL4, but were not present in the adult gland in the case of btIGAL4. prdmf9GAL4 presents a potentially useful GAL4 driver for further investigations of the binucleate cell formation in the accessory glands.
dc.format.extentix, 133 leaves.
dc.language.isoen_AU
dc.rightsAuthor retains copyright
dc.subject.lccQL537.D76 K43 2009
dc.subject.lcshDrosophila melanogaster Genetics
dc.subject.lcshDrosophila melanogaster Development
dc.subject.lcshDevelopmental biology
dc.titleUsing novel approaches to understand development: gene targeting, protein labelling and the development of the accessory glands in Drosophila melanogaster
dc.typeThesis (PhD)
local.description.notesThesis (Ph.D.)--Australian National University
dc.date.issued2009
local.type.statusAccepted Version
local.contributor.affiliationAustralian National University
local.identifier.doi10.25911/5d5fce9985497
dc.date.updated2018-11-20T23:07:47Z
dcterms.accessRightsOpen Access
local.mintdoimint
CollectionsOpen Access Theses

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