Perturbation of the orderly proliferation and differentiation of corneal epithelial cells can lead to corneal epithelial defects. Treatment of patients with a wide variety of corneal diseases (e.g., limbal deficiency of Stevens-Johnson syndrome, corneal alkali burn, neurotrophic keratopathy, recurrent erosion syndrome, corneal keratinization in ocular pemphigoid) is often compromised by an inadequate understanding of the regulation of corneal epithelial differentiation. The long term objective of this application is to provide a better understanding of corneal epithelial cell differentiation and normal corneal physiology for the design of a better treatment of corneal surface diseases. To achieve this long term objective we have developed anti-K12 keratin monoclonal and epitope-specific polyclonal antibodies, and cloned the murine cornea- specific keratin gene Krt1.12. The K12/K3 keratin pair forms the 10 nm intermediate filament in corneal epithelial cells and their expression signifies the cornea-type epithelial differentiation. It implicates that the Krt1.12 gene has unique regulatory DNA elements, and K12 keratin is essential for maintaining corneal epithelium integrity. To identify and characterize the cis-regulatory DNA elements (Specific Aim 1), we have prepared K12 promoter/enhancer-chloramphenicol acetyl transferase (CAT) DNA constructs. These reporter gene constructs will be used to transfect corneal and conjunctival epithelial cells in organotypic culture and to create transgenic mice. The cell-lineage-specific and tissue-specific expression of CAT activities will be determined to identify the cornea- specific cis-DNA elements. In vivo footprinting will be performed to further characterize the cornea-specific DNA elements. Using these cornea- specific DNA elements we will be able to design further experiments for targeted expression of other reporter genes, e.g. growth factors, receptors, integrins, etc. so that their roles in cornea can be elucidated. To determine the functional role of K12 keratin in the corneal epithelium (Specific Aim 2), we will create null K12 mutants via gene targeting technique to examine the effects of loss of K12 keratin on corneal epithelium and produce transgenic mice carrying defective murine Krt1.12 gene by injecting modified K12 genomic DNA into mouse fertilized eggs. The cornea epithelial phenotype of these transgenic mice will be examined. The corneal phenotypic changes of these transgenic mice will provide useful information in regard to clinical manifestations of human genetic defects involving Krt1.12 gene and to examine the hypothesis that structural mutation of Krt1.12 gene can lead to human cornea diseases.
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