The kidney filters blood through nephrons, to recycle water and solutes in order to maintain homeostasis in the body. A reduction in nephron formation during fetal development renders the affected individual susceptible to hypertension and end stage renal disease. Nephrogenesis relies on the iterative branching of the ureteric bud epithelium, which triggers the metanephric mesenchyme to condense and form nephrons at its tips. / A critical question during kidney development is to identify the genes that are responsible for nephron formation. The culture of mouse embryonic kidneys has been an important in vitro model to examine the effects of candidate genes during nephrogenesis. By culturing embryonic kidneys in the presence of growth factors, blocking antibodies to specific proteins, or by blocking transcription using RNA interference, gene function can be ascertained. Microinjection and electroporation has also been used to overexpress DNA constructs in mouse embryonic kidneys. We have reviewed the method of microinjection and determined the optimal conditions for introducing DNA into embryonic kidneys. We examined high voltage/short pulse parameters and compared them to previously reported low voltage/long pulse settings. The use of a high voltage with a short pulse led to 100 fold more transgene expression and a longer duration of expression, when compared to a low voltage/long pulse protocol. Furthermore, we can target either the ureteric bud or the mesenchyme of the developing kidney, by selectively microinjecting the transgene in the organ. / We have examined the expression pattern of Alk6 and members of the claudin gene family during kidney development. Their expression pattern along with their function has not been fully characterized in the embryonic kidney. In the future we plan to employ our novel electroporation protocol to study the function of these candidate genes in the developing kidney.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.97888 |
| Date | January 2005 |
| Creators | Alie, Tristan MacKay. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Human Genetics.) |
| Rights | © Tristan MacKay Alie, 2005 |
| Relation | alephsysno: 002331074, proquestno: AAIMR24597, Theses scanned by UMI/ProQuest. |
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