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K.K. H. Svoboda, J. Acevedo, P. Moessner; Electroporation and transfection of GFP–actin plasmids into whole embryonic corneal epithelia . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3791.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Whole embryonic corneal epithelial sheets can be isolated without the basal lamina. These sheets of cells respond to bombesin, lysophosphatidic acid (LPA) and extracellular matrix (ECM) molecules including fibronectin (FN), laminin and collagen (COL), by reorganizing the actin cortical mat (ACM). Green fluorescent proteins (GFP) have made it possible to study a wide variety of proteins in live cells. Fluorescent imaging of live cells enables researchers to visualize the cells to study cell–signaling pathways. Although transfection procedures for many cultured cells have been established, primary cells and whole tissues are difficult to transfect successfully. The objective of this research was to find an efficient method to transfect whole embryonic corneal epithelial tissues with a GFP–actin DNA plasmid in order to observe the effects of ECM stimulation on actin reorganization. Methods: Three different methods were used to transfect the primary corneal epithelial sheets. The first method was electroporation using gene transfer probes either in ovo or in vitro. Varying voltages (20–200V), varying times (50 µsec–75 msec) and varying pulses (1–2) were used to transfect tissues with GFP–actin (10 µg/ml). The second method used corneal epithelial sheets incubated overnight with liposomes produced in the laboratory (Reenstra et al., Investigative Ophthalmology and Visual Science, October, 2002) and the GFP plasmid (10 µg/ml). A third approach used Lipofectamine 2000 with Opti–Mem medium (Invitrogen). Varying concentrations of reagent (1–3 µl) and DNA plasmid (.8–1 µg) were tested. Results: The most effective results were obtained with Lipofectamine 2000 reagent, but rates of transfection did not exceed 50%. Electroporation was not as effective (25%) and lab made liposomes were the least effective (<25%). Conclusions: Whole epithelial sheets are difficult to transfect with plasmids. Other methods of transfection or infection with viral markers should be explored for higher rates of GFP expression.
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