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S. F. Geller, P. S. Ge, M. Visel, J. G. Flannery; A Single Lentiviral Vector for in vitro and in vivo Promoter Analysis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2917.
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© ARVO (1962-2015); The Authors (2016-present)
We have developed a lentiviral (LV) transfer vector plasmid (pFTMGW) to improve functional promoter analysis and transgene regulation in retinal gene therapy applications. Our goal was to identify cell-specific promoters to control and direct expression of therapeutic molecules in retinal cells. In the current study, we sought to validate modifications made to pFTMGW and assess its capability to direct Muller cell-specific gene expression in vivo.
We modified the pFUGW lentiviral transfer vector by adding a transcription blocking (TB) element immediately upstream of a novel multiple cloning site (MCS), thereby creating pFTMGW. We then cloned a 409 bp fragment (Vim409) of the SD rat vimentin gene into the pFTMGW vector. For in vitro evaluation of promoter activity, we transfected rat Muller cell cultures with 2 micrograms of both control and experimental plasmid DNAs, complexed with Lipofectamine 2000. Twenty-four hours later the cells were evaluated by microscopy, flow cytometry, and qRT-PCR. The Vim409 construct was packaged into lentiviral particles after in vitro validation experiments, and injected into the subretinal space of normal rats. In vivo expression was evaluated by fluorescent fundus examination. Rats were sacrificed, and processed for fluorescence microscopy five days after injection.
The pFTMGW construct allows for specific promoter regulated reporter gene expression while preventing undesired upstream CMV promoter function, which is required for LV vector packaging. The pFTMGW vector backbone exhibits very low intrinsic eGFP expression as measured by flow cytometry and qRT-PCR. The Vim409 plasmid drives robust eGFP reporter expression when transiently transfected into cultured Muller cells, at levels that approximate that of the ubiquitin-C and CMV promoters. The Vim409 LV virus infects and drives eGFP expression in cultured Muller cells, and directs expression exclusively in Muller cells when delivered to the retina in vivo.
The pFTMGW is a useful plasmid for testing of promoter elements. Combinatorial testing of cloned sequences is made possible by a synthetic MCS capable of accepting up to 5 regulatory elements. The plasmid greatly speeds the transition from in vitro to in vivo promoter testing by eliminating the need to shuttle regulatory elements between different vectors.
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