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N. Marsh–Armstrong, H.–W. Hwang, M. Steele, E. Callahan, M. Vetter; Early Retina Progenitor Specific Expression of Frizzled–5 BAC and Enhancer . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3240.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Frizzled–5 (FzD5) is expressed in early retina progenitors in both mouse and frog, and has been shown to regulate neural competence in the frog retina. We sought to define the key regulatory regions of FzD5 since this may enable the identification of factors that promote neural retina fates, may be used to drive expression of transgenes specifically in early retinal progenitors, and may enable the marking and purification of these cells from live animals. The Xenopus transgenic method is a rapid, inexpensive way for identifying functional regulatory regions in vertebrate genes. Methods:To tag the FzD5 gene, GFP was inserted into a 163kb bacterial artificial chromosome (BAC) containing the human FZD5 gene by homologous recombination in bacteria. Linearized BAC was used to make transgenic Xenopus laevis by the REMI/nuclear transfer method using conditions optimized for BAC transgenesis. To identify potential cis–regulatory regions within the BAC, we performed multiple species sequence alignments, and then tested 14 candidate regions between 1 and 92kb upstream of the FZD5 coding sequence for their ability to promote appropriate transgene expression. Results:The GFP–tagged human FzD5 BAC transgene was expressed in the early developing eye similar to the endogenous X. laevis FzD5 gene. A putative proximal promoter fragment, either alone or together with the first intron, did not drive similar expression. Multiple sequence alignments led to the identification of 14 candidate cis–regulatory regions. Of these, only one, located 44kb upstream of the FZD5 coding sequence, drove eye specific transgene expression, and did so irrespective of orientation. However, even after the removal of this cis–regulatory region, the GFP–tagged human FZD5 BAC retained eye–specific expression. Conclusions:Regulatory information governing gene expression in retina progenitors is conserved between mammals and amphibians. FzD5 regulatory regions drive expression in early retina progenitors and may be used to study various aspects of the biology of these important cells.
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