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J. T. Meij, E. C. Carlson, L. Wang, C.-Y. Liu, J. V. Jester, D. E. Birk, W. W. Kao; Cornea-Targeted Lumican Transgene Enhances Corneal Transparency and Keratocan Expression in Mice Lacking the Lumican Gene. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1485. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Keratocan and lumican are major structural proteoglycans in the vertebrate cornea. Both are synthesized and deposited by corneal stroma keratocytes. Mice lacking the gene for keratocan display a thinner, yet functionally normal cornea, whereas absence of lumican causes disarray of the stromal collagen matrix and ensuing corneal opacity. In addition, mice lacking the gene for lumican (LumKO) have reduced keratocan levels, consistent with our recent finding that lumican regulates keratocan transcription. In the present study, we investigated whether cornea-targeted overexpression of lumican rescued the LumKO phenotype.
LumKO mice were crossed with transgenic mice overexpressing lumican under control of the keratocan promoter (TG) and the off-spring corneas were examined by confocal microscopy through focusing (CMTF), electron microscopy, real-time PCR and SDS-PAGE/Western blotting (WB).
Scanning of LumKO/TG mouse eyes in vivo by CMTF showed similar epithelial and stromal thickness as in LumKO and WT mice. After digital image analysis of the CMTF scans for corneal light backscattering, a small but significant (P< 0.01) reduction in corneal haze was found in LumKO/TG (n=21) as compared to age-matched LumKO (n=13) mice. Subsequently, corneas from a subset of each group were processed for real-time PCR. Using ß-actin as reference gene, no significant differences were found at the keratocan mRNA level. In addition, one cornea per mouse was processed for WB and the relative levels of keratocan core protein were determined using Odyssey Infrared Imaging. On average, there was a ~2-fold increase in keratocan in LumKO/TG vs LumKO (P< 0.05), but the median values were similar. This reflects that in a few LumKO/TG the keratocan level was exceptionally high, whereas in most it was not above that in LumKO. Together these data suggest that, in LumKO cornea with low (initial) keratocan promoter activity, the expression of the lumican transgene is generally low; however, in some cases, lumican and keratocan expression may be subject to a feed-forward loop.
Expression of a lumican transgene in LumKO cornea is overall sufficient to improve corneal transparency.
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