Identification of the cornea-specific keratin 12 promoter by in vivo particle-mediated gene transfer

PURPOSE. Keratin 12 (K12) is a cornea epithelial cell-specific intermediate filament component. To provide a better understanding of its expression, i t is necessary to identify and characterize the promoter of Krt1.12 gene. METHODS. The 2.5-kb DNA 5' to Krt1.12 gene was sequenced. K12 promoter-Rho- gal DNA constructs were prepared and used in vivo to transfect rabbit corne as, conjunctivas, and skin by particle-mediated gene transfer (Gene Gun). I n vitro, the DNA constructs were transfected into cultured T-antigen-transf ormed rabbit corneal epithelial (RCE-T) cells and human fibrosarcoma HT-108 0 fibroblasts with lipofectamine. The promoter activity was assessed by mea suring beta-gal (beta-galactosidase) activity using histochemical staining with 5-Bromo-4-chloro-3-indolyl-beta-D-galactoside and enzyme assay with o- nitrophenyl beta-D-galactopyranoside. RESULTS. There are four Pax-6 pair box binding elements found between -910 and -2000 bp 5'-flanking the transcription initiation site of the Krt1.12 g ene. None of promoter constructs can be expressed by HT-1080 cells. Cotrans fection of Pax-6 cDNA with K12 promoter-beta-gal constructs containing Pax- 6 elements results in a fourfold increase of beta-gal activities in RCE-T c ells but not HT-1080 fibroblasts. The data of in vivo transfection in the r abbit by Gene Gun indicate that reporter gene constructs containing 0.6-kb and longer DNA fragments 5'-flanking Krt1.12 gene are effectively expressed in corneal, but not conjunctival or epidermal epithelial cells. CONCLUSIONS. The particle-mediated gene transfer is a suitable technique fo r in vivo delivery of transgenes to corneal epithelial cells. The 2.5-kb DN A fragment 5'-flanking Krt1.12 contains corneal epithelial cell-specific re gulatory cis-DNA elements. Pax-6 is a positive transcription factor essenti al for keratin 12 expression.