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EI Frolova, DC Beebe; Functional Analysis of the Promoter for the Long Isoform of Collagen a1(IX) Gene In Ovo . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2441.
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Purpose: We analyzed the promoter of the long-isoform of collagen α1(IX) gene to identify the elements responsible for ciliary epithelium-specific gene expression during the early stages of eye development. Methods: We extended the known promoter sequence using a promoter walking method. Several reporter constructs were generated that contained intron 1 and were different only in the length of the promoter fragment. We used green fluorescent protein (GFP) as a reporter and detected cells expressing the construct by confocal microscopy. To determine the function of intron 1 in regulation of expression, we replaced gene-specific intron 1 with intron 1 of the chicken ß actin gene. All constructs were evaluated by expression in chicken embryos using in ovo microelectroporation into the optic vesicle at stage 10 (E2). Results: All reporter constructs were expressed in the RPE and pigmented ciliary epithelium; only the longest construct was expressed in the non-pigmented ciliary epithelium. We previously showed that intron 1 was essential for expression in the eye. Replacing intron 1 of collagen α1(IX) gene with intron 1 of the chicken ß actin gene led to expression in all tissue of neural origin. We identified three short fragments in intron 1 that are highly conserved in the chicken, mouse and human genes. Conclusion: (1) Intron 1 contains regulatory elements that silence the transcription of the long-isoform of the collagen α1(IX) gene in the non-pigmented ciliary epithelium and retina. (2) The region located between -2120 and -1116 of the long-isoform of the collagen α1(IX) gene targets expression to the ciliary epithelium. (3) A basal promoter fragment of 67 nucleotides is sufficient to maintain reporter expression in the RPE and pigmented layer of ciliary epithelium in the presence of intron 1. (4) Different transcriptional control elements regulate the expression of the long-isoform of the collagen α1(IX) gene in the pigmented and non-pigmented ciliary epithelia.
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