Purchase this article with an account.
JC Blanks, J Pang, M Cheng, S Day, V Planelles; Photoreceptor Cell Rescue in Retinal Explants from Retinal Degenerative (rd) Mice by Gene Transfer using a Dicistronic Lentivirus Vector . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4597.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if lentivirus vector mediated ß-PDE delivery can rescue photoreceptor (PR) degeneration in retinal explants prepared from rd mice using a dicistronic construct, pHR-ß-PDE-IRES-GFP, that uses an internal ribosome entry site (IRES). The expression of ß-PDE gene, vital for PR rescue, was confirmed by anti-ß-PDE indirect immunofluorescence assay (IFA) in monolayers of transduced HeLa cells. IFA demonstrated complete correlation between the patterns of ß-PDE and GFP expression. Methods: Rd explants prepared on postnatal days (P) 6 were incubated with a lentiviral vector encoding both ß-PDE and GFP. Expression of ß-PDE is controlled by the CMV promoter and expression of GFP is facilitated by the presence of an IRES element placed directly upstream of GFP. This vector allows efficient expression of two open reading frames from a single promoter. After enucleation, each retina was isolated from the eyecup and RPE, placed on a Millipore insert and cultured in DMEM with 10% FCS. For each retinal expanlt, 30 µl of pHR-ß-PDE-IRES-GFP vector (6.6x106 infectious units/ml) was placed directly on the surface of the explant. Controls consisted of incubation in media without vector. After 24 hours, the explant was incubated in vector-free medium for 7 days in vitro (DIV). After 8 DIV, retinal explants were analyzed by light microscopy. The number of PR layers was determined in vector treated P6+8DIV explants compared to control explants and P14 rd retinas in vivo. Results: Fluorescence appeared around the edge of the explants 24 hours after vector treatment. GFP expression was apparent throughout the surface of the explant. No fluorescence was detected in controls. GFP+ cells were located primarily in the PR cells. There were approximately 8-10 layers of PR cells in vector treated retina compared with 5-7 in untreated controls. Areas of increased PR rescue corresponded to increased GFP expression. About 4 layers of PR cells were present in vivo in P14 rd mice. Conclusion: GFP is a valid surrogate marker for expression of ß-PDE in the pHR-ß-PDE-IRES-GFP vector. PR degeneration was delayed in untreated rd explants but not as much as in the (ß-PDE) treated retinas. Lentiviral mediated ß-PDE delivery rescued PR cells in rd retinal explant cultures prepared on P6 and incubated for 8 days. Work is in progress using this vector to extend PR rescue for a longer period in explants and by subretinal injections in rd mice.
This PDF is available to Subscribers Only