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Jing Xie, Bela Anand-Apte; Identification of Regulatory Sequences of Zebrafish Claudin 5 Genes. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1383.
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In our previous work, we have demonstrated that the blood-retinal barrier (BRB) is established in the hyaloid vasculature of zebrafish at 3 days postfertilization (dpf), and knock-down of claudin 5a gene results in leakage of plasma protein from the hyaloid vessels. The purpose of this study is to identify DNA regulatory sequences of the zebrafish claudin 5 genes.
The DNA regulatory sequences sufficient for the specific expression were identified through a transgenesis-based assay. DNA fragments of various lengths were amplified from the flanking regions of the claudin 5a or 5b, and inserted into the pT2KXIGΔin vector to replace the EF-1α promoter. The plasmids were co-injected with the Tol2 transposase mRNA into zebrafish embryos at 1- to 2-cell stage. From 2 to 5 dpf, the injected fish were sorted under a fluorescent microscope for EGFP expression in the hyaloid vessels.
The 5.6 kb fragment upstream of the start codon of claudin 5b can direct the EGFP expression in the hyaloid vessels from 2 dpf, but only in 2% of the injected embryos. A 2 kb sequence, from 6.5 to 8.5 kb upstream of claudin 5b, contains enhancer activities that can drive the expression in the retina and spinal cord.
The claudin 5b gene has positive and negative regulatory elements in the 5’ flanking region. The transgenic zebrafish line, cldn5:EGFP, is a new animal model and a powerful tool to study the BRB.
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