April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Identification of Regulatory Sequences of Zebrafish Claudin 5 Genes
Author Affiliations & Notes
  • Jing Xie
    Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
  • Bela Anand-Apte
    Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio
    Department of Cell Biology, Cleveland Clinic Lerner College of Medicine @CWRU, Cleveland, Ohio
  • Footnotes
    Commercial Relationships  Jing Xie, None; Bela Anand-Apte, None
  • Footnotes
    Support  This work was supported by US National Institute of Health EY016490, CA106415, EY015638, Research to Prevent Blindness (RPB) Challenge Grant, RPB Lew Wasserman award to BA-A., and Ohio BRTT 05.
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1383. doi:
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    • Get Citation

      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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      © ARVO (1962-2015); The Authors (2016-present)

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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 

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