Targeted expression of a lumican transgene rescues cornealdeficiencies in lumican-null mice

[摘要] Purpose: To investigate whether targeted expression of lumican inthe mouse cornea rescued the Lum-/- phenotype.Methods: Lum-/-/Kera-Lum mice were generated bycrossing Lum-/- mice with Kera-Lum transgenic mice thatoverexpressed lumican under the control of the keratocan promoter. Mouseeyes were analyzed in vivo by confocal microscopy through focusing(CMTF) to determine corneal sublayer thickness and haze. Subsequently,one cornea from each mouse was processed for SDS-PAGE/western blottingwhile the other was used for either electron microscopy (EM) orreal-time polymerase chain reaction (RT-PCR).Results: Overall, corneas of Lum-/-/Kera-Lum miceshowed significant improvement over Lum-/- but were stilldeficient when compared to wildtype (WT) mice. Specifically, analysis ofLum-/-/Kera-Lum mouse eyes by CMTF showed a similarstromal but slightly increased epithelial thickness compared to matchingLum-/- mice. Analysis of the CMTF scans for lightbackscattering revealed a small yet significant reduction in cornealhaze in Lum-/-/Kera-Lum mice as compared toLum-/- mice. At the EM level, the pronounced disarray of theposterior fibrillar matrix seen in Lum-/- mice was notobserved in Lum-/-/Kera-Lum mice. Moreover, analyses ofcollagen fibril diameter distributions showed a significant reduction inthe number of large-diameter (>40 nm) fibrils inLum-/-/Kera-Lum mice as compared to Lum-/- mice.No significant differences in keratocan expression were found at themRNA level, but western blot analysis detected an approximately two foldincrease in keratocan protein levels in Lum-/-/Kera-Lumover Lum-/- mice.Conclusions: Together these data suggest that despite the lowkeratocan promoter activity driving the transgene in Lum-/-cornea, transgenic lumican expression was sufficient to partially rescuecorneal phenotypic deficiencies.

[学科分类] 生物化学/生物物理