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V. Smyth, B.N. Kennedy; Identification of an Enhancer Within the 5’ Flanking Region of the Conetransducin A–Subunit . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3070.
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Purpose: The α–subunit of cone transducin (TαC) is expressed exclusively in cone photoreceptors both in the eye and pinealocytes in the pineal. TαC specificity makes it an ideal tool to identify cis–elements driving cone specific expression. This study examines the cis–elements responsible for the cell specific expression pattern of TαC in vivo. Methods:To identify cis–elements responsible for driving TαC expression, a series of deletions of the 5’ flanking region of the zebrafish TαC (zTαCP) gene were constructed upstream of the enhanced green fluorescent protein (EGFP) and tested in vivo. A transient assay was carried out by injecting these zTαCP–EGFP deletion reporter constructs, into one to two cell stage zebrafish embryos. Injected embryos were anaesthetised and analyzed by fluorescence microscopy at 5 days post fertilization (dpf). The zTαCP deletion constructs activity was measured by counting the number of GFP cells within embryos eyes at 5 dpf. Zebrafish embryos with greater than 50 GFP cells in the eye were scored as (+++), fish with 5–50 cells expressing GFP were scored as (++), 1–5 fluorescent cells as (+) and fish with no GFP were scored as (–). Results:A 1.2 Kb 5’ flanking region of zTαCP was initially identified as driving strong EGFP expression in cone photoreceptors. Deletion analysis of the 5’ flanking region refined this to a 200 base pair (bp) region and eventually to a 50 bp region. Significant loss of GFP expression is observed when this 50 bp enhancer region is deleted. Conclusions: A 0.05 Kb region of the zebrafish TαC 5’ flanking region acts as an enhancer. Progress in characterising this enhancer by electrophoretic mobility shift assay (EMSA) and the enhancer’s ability to drive heterologous promoters will also be presented. None
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