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Shambhu D Varma, Krish Chandrasekaran, ; Transfection of ARE-GFP Construct in Mouse Lens. Invest. Ophthalmol. Vis. Sci. 2014;55(13):724.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress plays a significant role in the pathogenesis of cataracts. While administration of nutritional antioxidants and metabolic agonists attenuates the process by scavenging the reactive oxygen species, the effect is short lived and highly variable due to constraints that limit their availability. Also, oxidative stress enhances transcription of several microRNAs that interfere with the biogenesis of transcription factors (Nrf2 and others) that regulate the transcription of antioxidant enzymes by binding with the AREs. Oxidative stress can affect this process through several routes. It was thus considered desirable to examine the feasibility of transfecting the lens with ingredients that influence the course of cataract development. Here, we examined the feasibility of using liposome incorporated ARE -GFP construct for such transfection studies.
Lenses from 6-week-old C57BL6J mice were incubated with the above construct incorporated in a liposome using Mirius 3D Transfection System. Briefly, the TransiT3D reagent was mixed with the construct in OptiMEM medium and incubated at room temperature for 15 min. It was then added to 1 ml of medium-199 containing the lens. The lens with the construct was then cultured for 24 hrs at 37 ° in an incubator gassed with 95% air and 5% CO2. Medium contained either 5mM glucose or 25mM galactose. The expression of GFP was monitored by fluorescence microscopy.
That the liposome penetrated the lens with its construct was highly apparent by the expression of ARE-GFP fusion protein, noticed by fluorescence in the lenses incubated with 5mM glucose as well as 25 mM galactose. It was also notable that fluorescence was significantly greater in the lenses incubated with 25mM galactose.
The results demonstrate that the liposome incorporated ARE- GFP DNA constructs can penetrate the intact lens. The construct also remained active, indicated by GFP florescence. The florescence was prominent in the equatorial region, the region of active cell differentiation. Also, it was noticeably greater in the galactosemic lenses, indicating a stress response. In summary, the results clearly indicate that the method can be used successfully to assess the functionality of the antioxidant system. Hence further studies are in progress to assess the transcription of ARE-GFP construct in the lens exposed to oxidative stress, as could be affected by purported anticataractogenic agents.
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