Transgenic rat models were used to investigate aspects of the role and expression of the circadian clock-regulated genes, vasopressin (VP) and Period 1 (Per1), in the suprachiasmatic nucleus (SCN), and the early growth response gene-1 (egr-1), in the SCN and anterior pituitary gland. Initial studies of the JP-17 and JP-59 (rat VP promoter / human growth hormone (hGH) reporter gene) transgenic lines detected expression of the reporter gene in the supraoptic nucleus (SON), but not the SCN, as determined by in situ hybridisation (ISH). Therefore, no further analysis of circadian clock-regulated VP expression could be conducted in these lines. Four (mPer1) promoter / destabilised enhanced green fluorescent protein (d2EGFP)) (Y) transgenic lines were generated, but failed to express the EGFP reporter gene. Sequence analysis of the transgene failed to explain the absence of expression. Subsequent studies in the egr-1 d4EGFP (57C) transgenic line detected tissue-specific constitutive expression and inducible expression in the brain, as determined by ISH. Region-specific and physiologically-regulated expression in the pituitary gland was also detected, as determined by Northern blot and dual ISH / immunohistochemical (IHC) analyses. Nevertheless, direct GFP fluorescence remained undetectable in this line. Seven egr-1/d2EGFP (Z) transgenic lines were then generated using a novel construct lacking the egr-1 intron present in the 57C construct. These transgenic lines demonstrated tissue-specific constitutive expression and inducible expression in the brain, and region-specific expression in the pituitary gland, at all levels of detection: transcript, protein and direct fluorescence. These findings indicate that the egr-1 intron is not required for directing tissue-specific and inducible expression in the rat. Z line rats will provide insights into the cellular progression of clock- and light- regulated responses in the brain. Potential uses of the egr-1/d2EGFP transgenic model are discussed
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:583417 |
| Date | January 2004 |
| Creators | Man, Pui Sin |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/55380/ |
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