Purchase this article with an account.
Zeynep Aktas, Sarah Slauson, B'Ann T Gabelt, Paul L Kaufman, Curtis R Brandt; Effect of proteasome inhibition on lentiviral vector transduction in human trabecular meshwork cells and monkey organ-cultured anterior segments.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5668.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate if proteosome inhibition using MG132 and actin cytoskeleton disruption with latrunculin-B increased the transduction efficiency of FIV vector co-expressing green flourescent protein (GFP) in human TM-1 cells and monkey organ-cultured anterior segments (MOCAS) and if this allowed for vector sparing.
TM-1 cells were plated in a 24-well plate at 1.5x104 cells/well. Cells were pretreated for 1 hr with DMSO or 5µm to 50µm MG132 or Latrunculin B (0.05µm, 0.1µm, 0.2µm), and transduced with FIV.GFP at a multiplicity of infection of 20. At 24 hours cells were fixed, stained with antibodies for GFP, and positive cells were counted. Cells transduced with FIV.GFP alone were used as controls. The effect of 20um MG132 treatment on high and low dose (2x10e7 and 0.8x10e7) FIV.GFP transduction was also tested in MOCAS and GFP expressions were graded. GFP expression in the TM was evaluated using fluorescence microscopy.
The percentage of GFP expression in FIV.GFP and DMSO+FIV.GFP groups were 24.2±4.6 and 25.2±4.1 (p=0.401). In the MG132 treatment groups the transduction was significantly increased at all concentrations. With latrunculin-B transduction efficiency was increased at all concentrations except in the group that received 0.2µm latrunculin-B. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20µM MG132 and high dose viral vector. However, grades of FIV.GFP expressions were comparable in eyes treated with 20µM MG132 and low dose viral vector.
MG132 increases the transduction efficiency of FIV.GFP in TM-1 cells and MOCAS. On the other hand, it does not seem to allow for vector sparing.
This PDF is available to Subscribers Only