Item type: | Article | ||||
---|---|---|---|---|---|
Journal or Publication Title: | Investigative Opthalmology & Visual Science | ||||
Publisher: | ASSOC RESEARCH VISION OPHTHALMOLOGY INC | ||||
Place of Publication: | ROCKVILLE | ||||
Volume: | 59 | ||||
Number of Issue or Book Chapter: | 1 | ||||
Page Range: | p. 298 | ||||
Date: | 2018 | ||||
Institutions: | Biology, Preclinical Medicine > Institut für Anatomie Biology, Preclinical Medicine > Institut für Anatomie > Lehrstuhl für Humananatomie und Embryologie Biology, Preclinical Medicine > Institut für Anatomie > Lehrstuhl für Humananatomie und Embryologie > Prof. Dr. Ernst Tamm | ||||
Identification Number: |
| ||||
Keywords: | PROTEIN-KINASE INHIBITOR; DOMINANT-NEGATIVE RHOA; OUTFLOW FACILITY; GENE-TRANSFER; TRANSGENE EXPRESSION; INTRAOCULAR-PRESSURE; UVEOSCLERAL OUTFLOW; CULTURED HUMAN; OPHTHALMIC SOLUTION; ANTERIOR SEGMENTS; trabecular meshwork; FIV vector; MG132; gene therapy proteasomes | ||||
Dewey Decimal Classification: | 500 Science > 570 Life sciences | ||||
Status: | Published | ||||
Refereed: | Yes, this version has been refereed | ||||
Created at the University of Regensburg: | Yes | ||||
Item ID: | 47681 |
Abstract
PURPOSE: To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS). METHODS: TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 mu M MG132 and transduced with FIV.GFP (green fluorescent protein)- or ...
Abstract
PURPOSE: To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS). METHODS: TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 mu M MG132 and transduced with FIV.GFP (green fluorescent protein)- or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 mu M MG132 treatment on high- and low-dose (2 x 10(7) and 0.8 x 10(7) transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA. RESULTS: In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 mu M MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus. CONCLUSIONS: Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.
Metadata last modified: 28 Jul 2021 17:28