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S.F. Geller, P.S. Ge, N. Rahmanattar, M. Visel, K.P. Greenberg, J.G. Flannery; Generation and Testing of a Lentiviral Vector for in vitro and in vivo Analysis of Cell Specific Promoters . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4908.
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© ARVO (1962-2015); The Authors (2016-present)
To generate a lentiviral (LV) backbone vector for gene promoter analysis in live animals and cultured cells, and to test the capacity of short (less than 1500 bp) evolutionary conserved regions (ECRs) of non–coding DNA sequences to direct gene expression in Müller cells.
Our LV vector (MT2) includes 2 tandem 154 bp transcription blocking (TB) sequences and a novel multiple cloning site (MCS) upstream of enhanced green fluorescent protein (EGFP). SYBR Green real–time RT–PCR was used to assess the ability of the TB sequences to mitigate EGFP expression from an upstream CMV promoter required for viral production. ECR sequences were analyzed and chosen using bioinformatic programs at www.dcode.org. Highly conserved fragments (at least 70% sequence identity using a 100 bp window across 2 or more species) were amplified by PCR using c57BL/6 mouse or SD rat genomic DNAs, and were cloned into MT2. Plasmid DNA transfected into cultured rat Müller cells using Lipofectamine 2000. Cultured cells were processed for total RNA extraction and RT–PCR, or, following formaldehyde fixation, mounted on microscope slides and digitally photographed using a green fluorescence filter.
TB sequences reduced EGFP expression by 92% and 94% with 1 and 2 TBs, respectively. Virtually undetectable levels of EGFP expression are observed in vectors lacking an MCS–cloned promoter. Short ECRs of two Müller cell expressed genes, CD44 and vimentin, were cloned into our modified vector (MT2). Of an initial pool of 30 ECRs, several fragments demonstrated an ability to drive expression of EGFP, 2 of which led to robust expression and represented DNA sequences immediately upstream of the first coding exon of the vimentin gene in both mouse and rat. These data suggest that a short (approximately 325 bp) region of the rodent vimentin promoter immediately upstream of exon 1 is capable of directing gene expression in Müller cells.
We sought to develop a pluripotent vector that would obviate the need for re–cloning promoters into multiple vectors used in diverse experiments such as transfection, electroporation, virus production and infection, and transgenic animal generation. We succeeded in generating a lentiviral vector useful for testing and evaluating promoter activity in cultured cells. This vector promises to expedite promoter testing in numerous laboratory assays, including the use of active viruses, both in vitro and in vivo.
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