Abstract
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). We have previously shown that Rho and ROCK are necessary for actin reorganization in corneal epithelial sheets. The objective of this research was to produce an YFP–Rho vector and express it in primary epithelial and fibroblast monolayers cultures and whole epithelial sheets.
Methods: :
Chicken RhoA RNA was isolated from corneal epithelia extracts, and cloned into an eYFP–N1 plasmid vector (BD Biosciences). The plasmid was transfected into COS and 3T3 cells using FuGENE to determine the optimal concentrations. Primary chicken corneal fibroblast and epithelial cells were isolated and grown in monolayers cultures and whole corneal epithelial sheets were isolated from E8 chicks. Varying concentrations of FuGENE (1.5–4.5 µl) and DNA plasmid (.5–1 µg) were tested. Epithelial sheets were stimulated with fibronectin, collagen or lysophosphatidic acid to determine if the YFP–Rho changed intracellular distribution. Cells were counter stained with phalloidin or a nuclear stain (ToPro).
Results: :
FuGENE did not appear to adversely affect cell morphology. All transfected cells expressed YFP–Rho including the corneal epithelial cells maintained in sheets. The transfected cells responed to ECM proteins.
Conclusions: :
FuGENE was successful as a transfection agent in both primary and cell line monolayer cultures and whole corneal epithelial sheets. This YFP construct may be useful in future studies analyzing Rho translocation in living cells and epithelial sheet organ cultures.
Keywords: cornea: basic science • signal transduction • cytoskeleton