The functional analysis of the ocean pout (Macrozoarces americanus) type III antifreeze protein gene promoter

Kirby, Trina Maxine (2005) The functional analysis of the ocean pout (Macrozoarces americanus) type III antifreeze protein gene promoter. Masters thesis, Memorial University of Newfoundland.

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Abstract

The full length ocean pout antifreeze gene promoter and a series of promoter deletion constructs were fused to the luciferase reporter gene, and their transcriptional role was examined in a transient expression assay by transfection into three cell lines; a human epithelial (CRL-1578), a human fibroblast (HTB-92) and a Chinook salmon embryonic cell line (CRL-1691). The studies demonstrate that the ocean pout AFP gene promoter can function in both mammalian and fish cell lines and that they contain both positive as well as negative regulatory regions. This indicates that the AFP gene may be regulated at various sites. -- In previous studies, a fish antifreeze/ growth hormone chimeric gene construct opAFP-GHc2 was injected into Atlantic salmon eggs. Through PCR analysis it was found that this construct reorganized in the fish genome so that the beginning of the 5’ ocean pout AFP promoter end was linked to the 3' end of the AFP polyadenylation sequence. In my study, this reorganized transgenic promoter was also ligated to the luciferase reporter gene and examined in a transient transfection assay, which determined that this reorganized promoter had significantly less activity than that of the full-length intact promoter. However, promoter activity was detected in all three cell lines which indicates that the transgene should be expected to express growth hormone in vivo.

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