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M. S. Hansen, N. Singh, V. Jessop, S. Moisyadi, B. Ambati; Treatment of Corneal Neovascularization Through Transposase-Mediated Somatic Insertional Mutagenesis for Long-Term Expression of Flt2-3k. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4958.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether, FLT2-3K intraceptor, a recombinant construct of domains 2 to 3 of VEGFR-1 (Flt) coupled with an endoplasmic reticulum retention signal (KDEL), can be expressed for long periods using somatic integration.
In order to attain long-term expression of FLT2-3K intraceptor, that sequesters VEGF, we have utilized the insertional property of the piggybac transposase. Transposases are enzymes with the ability of mobilizing DNA via a cut-and-paste mechanism. Transposon-mediated mutagenesis has largely been reserved for genetic manipulating of lower organisms because of low integration rates and nonspecific integration, which can lead to undesirable consequences. Piggybac has been shown to have the highest integration efficiencies compared to other transposases. We generated a fusion protein using the piggybac vector that will express FLT2-3K intraceptor for long periods using non-viral somatic genomic integration.
Preliminary results in vitro showed that the piggybac mediated expression protein proved to be a successful system for specific insertional mutagenesis. Integration of desired DNA was determined by visualization of GFP within the cells.
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