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L.K. Yeh, J. Ouyang, C.–Y. Liu, W.–Y. Kao, I.–J. Wang; Molecular Analysis of the Zebra Fish Lumican (zLum) and Keratocan (zKera) Genes . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2610.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To establish a new vertebrate model system for studying molecular genetics of the corneal diseases in zebrafish. Methods:In the present study, we isolated and identified the zebrafish corneal keratan sulfate proteoglycan genes, zLum and zKera. Human lumican and keratocan sequences were used to blast search zebrafish homologues. Oligo–primers specific to the zLum and zKera were synthesized for polymerase chain reaction (PCR). The zLum and zKera full–length genomic DNA and cDNA were isolated by PCR of genomic DNA and RT–PCR of total eye RNA, respectively. The structures of the zLum and zKera gene were determined by Southern blot hybridization, sequencing, and DNA analysis tools provided by DNASIS MAXTM. Northern blotting and in situ hybridizations were carried out to determine the expression patterns of zLum and zKera. Results:Genomic southern blotting analysis showed that zebrafish genome contains single copy of zLum and zKera genes. In zebrafish genome, the zLum and zKera genes are 11 kb apart where zLum is located 5’upstream to the zKera. The zLum and zKera span 3.0 kb and 2.2 kb of the zebrafish genome, respectively. Both genes consist of 3 exons and two introns, and do not have a conventional TATA box of transcription initiation in the 5’–flanking regions. zLum and zKera mRNA encode 344 and 341 amino acids as verified by cDNA sequence. The zebrafish lumican and keratocan are 50% and 56% identical in amino acid sequence to their counter parts of human proteins. Northern hybridization revealed that zLum mRNA was detected in many tissues including eye, eyeless head, and headless body, while zKera mRNA was only found in the eye. In situ hybridization confirmed that zKera mRNA was restricted to corneal stroma, while zLum mRNA was present in corneal and scleral stroma. Conclusions: These results demonstrate that lumican and keratocan gene structure and expression patterns are highly conserved in zebrafish and human. Thus, zebrafish may serve as a model to studying corneal biology and diseases.
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