December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Generation of the Keratocan Gene-Targeted Mutagenesis in the Mouse
Author Affiliations & Notes
  • C-Y Liu
    Department of Ophthalmology
    University of Cincinnati Cincinnati OH
  • JR Hassel
    Shriners Hospitals for Children Tampa FL
  • B Kane
    Shriners Hospitals for Children Tampa FL
  • TC Doetschman
    Molecular Genetics
    University of Cincinnati Cincinnati OH
  • WW Y Kao
    Department of Ophthalmology
    University of Cincinnati Cincinnati OH
  • Footnotes
    Commercial Relationships   C. Liu, None; J.R. Hassel, None; B. Kane, None; T.C. Doetschman, None; W.W.Y. Kao, None. Grant Identification: NIH Grant EY12486
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 2936. doi:
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    • Get Citation

      C-Y Liu, JR Hassel, B Kane, TC Doetschman, WW Y Kao; Generation of the Keratocan Gene-Targeted Mutagenesis in the Mouse . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2936.

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

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Abstract: : Purpose: Keratocan (Kera) belongs to the small leucine-rich proteoglycan (SLRP) gene family. It is one of the major components of extracellular keratan sulfate proteoglycan (KSPG) in vertebrate corneal stroma. During embryonic development, Kera gene expression tracks ocular surface tissues morphogenesis including cornea and eyelids. It has been suggested that corneal KSPGs play a pivotal role in interacting specialized collagens to produce transparent cornea. In human, mutations in the KERA gene are associated with cornea plana; the forward convex curvature is flattened, leading to a decrease in refraction. In order to investigate the biological role of the Kera gene and to establish an animal model for corneal plana, we generated Kera knockout mice via gene targeting. Methods: A 1.4 kb fragment from the Xho I site in intron 1 to a Kpn I site in exon 2 was deleted and replaced with a positive selection marker, neomycin resistance gene (pgkpr-neopA) cassette in the antisense orientation with respect to the Kera gene. A negative selection marker gene, diphtheria toxin A fragment (pgkpr-Dta) cassette, was placed on the 3' end of the targeting vector. The final targeting vector, Kera-TV, contains a 3.1 kb and a 1.5 kb homologous sequences on 5' and 3' end, respectively. PCR and Southern blotting analysis were used to identify targeted allele. Northern, western blotting and immunohistochemical analysis were performed to characterize the expression of Kera mRNA and protein expression, respectively. Finally, histological analysis was done using light microscopy. Results: Northern , western blotting and imuunohistochemical analysis showed that no Kera mRNA or keratocan protein was detected in the Kera-/- cornea. Mice with heterozygote of the Kera mutation (Kera+/-) appeared normal transparent cornea. Up-to-date, mice with homozygote of the Kera mutation (Kera-/-) have not yet appeared hazy cornea at the age of 4 weeks, however, the corneal stroma is 30% thinner than that of the wild-type littermate. Conclusions: Our results suggest that that Kera-/- resulted in keratocan-null mutant. Detailed analysis of the phenotypic changes of cornea in the older Kera-/- mice will be performed. It is anticipated that the generation of Kera-null mice can uncover the function of keratocan in cornea development and can serve as a model to study the pathogenesis of corneal plana.

Keywords: 316 animal model • 370 cornea: basic science • 374 cornea: stroma and keratocytes 

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