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S. Yoshikawa, E.G. Norcom, R.W. Yee, X.C. Zhao; Promoter/Enhancer Analysis of the Zebrafish gelsolin–like 1 (gsnl1) Gene for the Cornea Specific Expression . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1582.
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© ARVO (1962-2015); The Authors (2016-present)
Our long–term goals are to use zebrafish as an animal model to study human corneal diseases. Cornea–specific promoters are powerful genetic tools for the analysis of corneal disease genes. The zebrafish gsnl1 gene encodes an actin filament regulatory protein and is specifically expressed in the zebrafish cornea. The purpose of this study is to clone and analyze the cornea–specific promoter of gsnl1.
A 6.5–kb genomic DNA fragment located on the upstream of the transcription initiation site of gsnl1 was cloned into a GFP expression vector. Several clones were generated by deleting the 5’ region of that fragment. The function of the promoter was studied by transgenic analysis. The plasmid was microinjected to 1–2 cell stage embryos and the transient GFP expression was examined by fluorescence microscopy on embryos of 1–3 day post fertilization (dpf).
DNA constructs containing 6.5–kb and 2.5–kb fragments of the putative promoters produced a specific GFP expression on the surface of the embryo including the corneal epithelial cells and in the notochord. The corneal expression was confirmed on frozen sections. At 1dpf, 86% and 55% injected embryos displayed strong GFP signals on the surface for the plasmids containing 6.5–kb and 2.5–kb fragment, respectively. Removal of the 1–kb region from the 2.5–kb fragment severely diminished the promoter activity. Deletion of the intron region from the 2.5–kb fragment also greatly reduced the number of the GFP expressing cells.
We have cloned the 5’ upstream region of the zebrafish gsnl1 gene. The transgenic analysis indicated that the putative promoter region contained the functional promoter. The DNA fragment located between 2.5–1.5 kb upstream of the translation initiation site was found necessary for the specific expression of GFP on the surface ectoderm including the corneal epithelial cells. This region may contain the cell–type specific cis–regulatory element(s). The first intron was also found to have an enhancer activity. Since the GFP expression on the non–corneal ectoderm and notochord is ectopic, compared to the normal expression pattern of the gsnl1 gene, there should be some negative regulatory element(s) in the rest of the gene.
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