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Mason Posner, Zachary Haley, Amy Drossman; Assessing the zebrafish as an efficient model for analyzing mammalian α-crystallin transcriptional regulation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5741.
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© ARVO (1962-2015); The Authors (2016-present)
The regulation of α-crystallin expression is of interest due to the role that these small heat shock proteins play in cellular stress response and their involvement in the etiology of diverse diseases. In this study we tested the hypothesis that zebrafish embryos can be used as an efficient model system for assessing the regulatory activity of mammalian αA- and αB-crystallin promoter sequences.
Zebrafish and mouse genomic sequences were used to design nucleotide primers for amplification of varying lengths of putative promoter regions for αA- and αB-crystallin. Zebrafish genomic DNA and mouse BAC clones were used as templates for amplification reactions and resulting products were ligated into the pAcGFP1-1 vector upstream of the gene encoding green fluorescent protein (GFP). Purified plasmids were injected into 1-cell stage zebrafish embryos and GFP expression was monitored by fluorescence microscopy.
Injection of a combined mouse αA-crystallin promoter and enhancer region (-111 to +46 and DCR1), known to drive expression in the mouse lens, produced lens specific GFP expression in zebrafish embryos. Comparison of the genomic sequences upstream of mouse αB-crystallin and zebrafish αBb-crystallin showed a similar tail-to-tail arrangement with the small heat shock protein Hspb2. The region between these two genes in the zebrafish is 5 kb, much larger than the 1 kb found in the mouse. A construct containing the 2 kb upstream of the zebrafish αBb-crystallin gene drove expression of GFP in the zebrafish lens and skeletal muscle, replicating native expression. However, injection of either a 0.8 or 1.4 kb mouse αB-crystallin promoter construct did not result in zebrafish lens or skeletal muscle expression, but did drive GFP expression in the notochord.
These results show that mouse αA-crystallin promoter activity can be replicated in zebrafish embryos, suggesting that the zebrafish can be used as an efficient model for screening modified mammalian promoter constructs. However, while the genomic organization of mouse αB- and zebrafish αBb-crystallin is evolutionarily conserved, their gene regulatory mechanisms have apparently diverged. Further examination of mouse and zebrafish αB-crystallin promoters, including the zebrafish lens-specific αBa-crystallin, can provide insights into the evolution of altered tissue specificity in this protein family.
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