June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Knock-out of Hsp27 in Danio rerio
Author Affiliations & Notes
  • Smriti Mishra
    Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee, United States
  • Sanjay Mishra
    Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee, United States
  • Alexandra Fuller
    Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee, United States
  • SHUYU WU
    Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee, United States
  • Hassane S Mchaourab
    Molecular Physiology & Biophysics, Vanderbilt University, Nashville, Tennessee, United States
  • Footnotes
    Commercial Relationships   Smriti Mishra, None; Sanjay Mishra, None; Alexandra Fuller, None; SHUYU WU, None; Hassane Mchaourab, None
  • Footnotes
    Support  NIH Grant EY12018
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5306. doi:
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    • Get Citation

      Smriti Mishra, Sanjay Mishra, Alexandra Fuller, SHUYU WU, Hassane S Mchaourab; Knock-out of Hsp27 in Danio rerio . Invest. Ophthalmol. Vis. Sci. 2017;58(8):5306.

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

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Abstract

Purpose : Small heat shock proteins (sHSP) including αA, αB and Hsp27 play important role in proteostasis although the exact role of sHSP in maintenance of the optical transparency of the lens is not completely established in vivo. Hsp27 is expressed in lens, retina and cornea besides heart and neurons. It is upregulated under various stresses including age-related macular degeneration (AMD) and ocular cancers. To understand the role of Hsp27 in diseases, we have created knock-out (KO) zebrafish (Danio rerio) lines using CRISPR-Cas9 system that disrupts the hspb1 (hsp27) gene. We have also cloned Hsp27 and studied its chaperone activity in vitro.

Methods : Zebrafish larvae (3-5 dpf) treated with 1-phenyl 2-thiourea (PTU) were examined by bright field (BF) and differential interference contrast (DIC) microscopy for screening the phenotypes. Danio Hsp27 protein was purified from E. coli expression system and assayed for binding to destabilized mutants of T4 Lysozyme. We also used multi-angle light scattering to estimate the oligomeric size.

Results : We targeted hspb1 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 insertions and deletions (INDELS) and the presumptive null alleles, confirmed by sequencing are propagated to subsequent generations. Danio Hsp27 forms oligomers larger than human Hsp27 and shows chaperone-like activity against fluorescently labeled model substrate T4-lysozyme.

Conclusions : Although preliminary, our results suggest that Zebrafish can be used as a model to study the role of Hsp27 in various proteostatic diseases. The transparency and relatively large eye lens make zebrafish embryos an ideal model to visualize alterations in molecular interactions induced by genetic manipulations of sHSP.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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