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P. Ge, S. F. Geller, M. L. Chow, M. Visel, J. G. Flannery; Comparative Assessment of Retinal Glial Promoters. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3781. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Targeting expression of therapeutic molecules to retinal Müller cells is hampered by the lack of regulatory elements suitable for this purpose. We sought to use comparative genomic approaches to identify functional regulatory sequences that can direct Müller cell-specific expression of transduced genes.
We modified a lentiviral transfer vector (pFTMGW) with a novel multiple cloning site (MCSv2) to generate a new vector, pFTM3GW. Using a suite of bioinformatics software at www.dcode.org, we identified and cloned a full-length promoter (1500bp), a proximal promoter (500bp), and an evolutionarily conserved region (ECR; at least 70% sequence identity over 100 bases between human and mouse genomes) for each of 10 known Müller cell expressed genes, including: GFAP, Carbonic Anhydrase, s100ß, Glutamine Synthetase, GLAST, mGluR6, CD44, Vimentin, CRALBP, and PDGFRα. Each ECR (average length of approximately 300 bp) lies immediately proximal to the transcriptional start site. All sequences were PCR amplified from C57BL/6J mouse genomic DNA, cloned into the pFTM3GW vector, and transfected into cultured rat Müller cells. Expression of the eGFP reporter was evaluated by fluorescence microscopy, flow cytometry, and quantitative RT-PCR.
We cloned ~60 potential regulatory elements from 10 Müller cell expressed genes. The cloned sequences drove eGFP expression in cultured Müller cells at variable levels. Our results suggest that inclusion of untranslated regions (UTR) in cloned promoter elements correlates with and thus may be important for high-level eGFP expression. Conserved transcription factor binding site (TFBS) analysis implicates the cell’s utilization of short, conserved sequences for controlling gene expression.
pFTM3GW is a versatile lentiviral vector transfer plasmid for use in transfection assays. We demonstrate that different Müller cell specific promoters possess the inherent potential to direct transgene expression at unique levels. Further analysis should aid in the identification of transcription factor binding sites and elucidate their contribution to cell-specificity and gene expression levels. The use of these regulatory elements should improve our ability to regulate therapeutic gene expression in gene therapy applications.
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