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Development of an in vitro model for analysis of transgene expression in the hen oviduct

An <i>in vitro</i> method in which to investigate transgene expression in the chicken oviduct prior to the generation of transgenics is desirable. The egg white protein genes are expressed in the tubular gland cells (TGCs) of the oviduct of laying hens. TGCs were isolated from the magnum region of the oviduct of adult hens and were found to be sufficiently viable in culture for use to investigate transgene expression. Egg white mRNAs and proteins were detected in TGCs maintained <i>in vitro</i> for 72 hours but tests of transfection using several commercially available reagents, electroporation and lentiviral transduction indicated that the frequency of gene transfer was too low to be useful. As an alternative, the use of oviduct tissue explants was investigated. Explants were isolated from the magnum region of the oviduct of sexually mature laying hens and the cells in the explants were found to be sufficiently viable in culture for use to investigate transgene expression. Egg white mRNAs and proteins were detected in the explants maintained <i>in vitro</i> for 4-5 days. Transfection using reagents was not successful but gene transfer into the explants was successfully achieved through electroporation. Expression of a therapeutic protein was detected in the explants after electroporation with a transgene constructs carrying a ubiquitous promoter or the ovalbumin promoter. Gene transfer into the explants was also achieved through transduction with EIAV and HIV vectors pseudotyped with a variety of envelope proteins. The highest levels of transduction were achieved using vectors pseudotyped with the vesicular stomatitis virus G envelope protein. Expression of a therapeutic protein was detected in the explants after transduction with viral vectors carrying a ubiquitous promoter or the ovalbumin promoter. Chicken oviduct explants were identified as suitable for use <i>in vitro</i> to investigate transgene expression in the chicken oviduct.

Identiferoai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:652730
Date January 2005
CreatorsHunter, Cheryl Victoria
PublisherUniversity of Edinburgh
Source SetsEthos UK
Detected LanguageEnglish
TypeElectronic Thesis or Dissertation
Sourcehttp://hdl.handle.net/1842/15062

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