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L.–K. Yeh, C.–Y. Liu, W.–Y. Kao, C.–L. Chien, I.–J. Wang; Molecular Characterizations of the Zebrafish Lumican (zLum) and Keratocan (zKera) Genes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3006.
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© ARVO (1962-2015); The Authors (2016-present)
In the present study, we aimed to isolate zebrafish lumican (zLum) and keratocan (zKera) genes and to develop a zebrafish model of corneal development.
The zLum and zKera full–length genomic DNA and cDNA were isolated by PCR of genomic DNA and RT–PCR of total RNA from eye, respectively. The structures of the zLum and zKera genes were determined by Southern blot hybridization, DNA sequence analysis by DNASIS MAXTM. Northern blotting and in situ hybridizations were carried out to determine the expression patterns of zLum and zKera. A green fluorescent protein (GFP) cDNA driven by zLum or zKera promoter was used to produce transgenic zebrafish lines by microinjection.
Both zLum and zKera appear as single copy genes in the zebrafish genome. The zKera gene is located 11 kb downstream from zLum. The cDNA analysis predicted that open reading frame of the zLum and zKera encoded 344 and 341 amino acids, respectively. The zLum and zKera shared 50% and 56% identity in amino acid sequence to their human counter parts. Northern blotting hybridization revealed that zLum mRNA was about 2.0 kb in size and expressed in many tissues including eye, brain, muscle and fin. In contrast, the zKera mRNA was ∼4 kb and detected only in the eye but not in other tissues of the adult fish. In situ hybridization confirmed that zKera mRNA was restricted to cornea, while zLum mRNA was present in eyes and other tissues. Moreover, the zLum promoter–GFP drove GFP expression in various tissues including eyes, while zKera promoter–GFP transgene revealed cornea–specific expression of the GFP in adult transgenic zebrafish.
The data indicated that mammalian corneal SLRP genes i.e., lumican and keratocan, are highly conserved in zebrafish at the level of gene structure, expression patterns, and promoter activities. Therefore, zebrafish may be used as a new vertebrate model to studying biology and molecular genetics of corneal development and diseases.
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