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J. Xie, E. Farage, B. Anand-Apte; A New Transgenic Zebrafish Model for Studying the Blood-Retinal Barrier. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2939.
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To develop an in vivo model for studying the blood-retinal barrier, by generating a transgenic zebrafish line expressing a vitamin D-binding protein fused with enhanced green fluorescent protein (DBP-EGFP), as an endogenous tracer in the blood plasma.
Fluorescein isothiocyanate-dextran 4 (FD4) was injected into the sinus venosus of zebrafish embryos and the leakage was evaluated by confocal microscopy. The endothelial BRB structure in zebrafish was demonstrated by immunohistochemical staining of three tight junction markers, claudin-5, ZO-1 and occludin. Expression of the DBP-EGFP was driven by the hepatocyte-specific promoter of the liver-type fatty acid binging protein (L-FABP). The transgenic larvae were treated with bradykinin (BK), and the DBP-EGFP leakage out of the retinal blood vessels was identified by fluorescent microscopy.
Expression of claudin-5, ZO-1 and occludin was observed in the retinal vasculature at a relatively high level from 3 dpf (days post fertilization) at which stage the blood retinal barrier appears to be established. The transgenic fish expressed the DBP-EGFP specifically in the blood plasma. Treatment with 0.1 mM BK resulted in leaking of DBP-EGFP out of the retinal blood vessels in about 30% of the fish.
The development of the zebrafish endothelial BRB structure occurs at 3dpf. The Tg (l-fabp:DBP-EGFP) fish is a new animal model that can display the BRB, which will have great advantages in performing forward-genetic screens to identify the genes involved in the formation and maintenance of the BRB.
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