June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Zebrafish Models of αB1-Crystallin to Study Human Cataract
Author Affiliations & Notes
  • Sanjay Mishra
    Chemical & Physical Biology Program, Vanderbilt University, Nashville, TN
    Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN
  • Ping Zou
    Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN
  • Simon Wu
    Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN
  • Hanane A Koteiche
    Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN
  • Poonam Rathore
    Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN
  • Wenbiao Chen
    Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN
  • Kevin L Schey
    Department of Biochemistry, Vanderbilt University Medical Center, Nashville, TN
  • Hassane S Mchaourab
    Department of Molecular Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN
  • Footnotes
    Commercial Relationships Sanjay Mishra, None; Ping Zou, None; Simon Wu, None; Hanane Koteiche, None; Poonam Rathore, None; Wenbiao Chen, None; Kevin Schey, None; Hassane Mchaourab, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5878. doi:
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      Sanjay Mishra, Ping Zou, Simon Wu, Hanane A Koteiche, Poonam Rathore, Wenbiao Chen, Kevin L Schey, Hassane S Mchaourab; Zebrafish Models of αB1-Crystallin to Study Human Cataract. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5878.

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

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Abstract

Purpose: Several αB-crystallin mutants have been implicated in autosomal cataracts in humans. While studies in vitro have demonstrated abnormal chaperone activity in these mutants, the exact role of αB in maintenance of the lens transparency is not established in vivo. Zebrafish (Danio rerio) is an attractive vertebrate model system to elucidate the role of αB-crystallin because it expresses a lens specific constitutively active αB1 and a ubiquitous αB2 paralog. We are in process of creating stable knock-out (KO) zebrafish lines using CRISPR-Cas9 system that disrupts the αB1 gene (cryaba). We have also generated zebrafish that express pathological human αB mutant R120G. We demonstrate that αB1 knock-out in zebrafish leads to lens phenotypes that can be used to monitor etiology of cataract.

Methods: Zebrafish larvae (3-5 dpf) and adults were examined by bright field (BF) and differential interference contrast (DIC) microscopy for screening the lens phenotype.

Results: We targeted cryaba by co-injecting custom guide RNA and the zebrafish codon-optimized Streptococcus pyogenes Cas9 RNA into one-cell-stage AB embryos. The adult founders were screened for germ-line transmission of small insertions and deletions (INDELS) in cryaba and the presumptive null alleles, confirmed by sequencing are maintained. We also constructed Tol2 transposon-based transgene that expresses the cDNA of the mutant human αB-crystallin R120G under the control of zebrafish αA-crystallin promoter. The transgene also included the reporter (cmlc2:mCerulean) to fluorescently mark cardiac myocytes for identification of insertion. The transgene was co-injected with Tol2 transposase RNA. The fish with fluorescent heart were confirmed for desired genotype by sequencing and are maintained as stable transgenic lines. Preliminary analysis reveals cataract-like phenotypes in the embryos of CRISPR-Cas9 KO of αB1; and both in the embryos and adults of transgenics expressing the mutant αB1-R120G protein. In addition, we have begun to use MALDI mass spectrometry to monitor change in the proteome and lipid profiles of cataractous zebrafish lenses.

Conclusions: Although preliminary, our results suggest that Zebrafish can be used as a model for hereditary cataracts. The transparency and relatively large eye lens make zebrafish embryos an ideal model to visualize alterations in molecular interactions induced by genetic manipulations of α-Crystallins.

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