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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)
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.
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.
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.
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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