Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: retinal development