Abstract
Abstract: :
Purpose: It has been previously demonstrated that a 3.2 kb DNA fragment up–stream of keratocan gene (Kera) has the promoter activities for the tissue specific expression of reporter genes similar to the expression pattern of Kera (Liu et al, Gene, 250: 85–96, 2000). The purpose of present study was to identify the cis–regulatory DNA elements modulating Kera expression. Methods: Deletion mutant reporter genes containing 0.3, 1.5, 2.6 kb DNA fragments 5’ to Kera gene were prepared from the Kerapr–3.2kb–ßGeo minigene. The functionality of the reporter minigenes were determined by measuring ß–galactosidase (ß–gal) activities in tissues of transgenic mice produced by microinjection, and the injection of minigenes into corneal stroma and skeletal muscles of wild type mice. Results: Only transgenic mice carrying the 3.2 kb Kera promoter exhibited ß–gal activity in a pattern similar to what was previously reported, except the enzyme activity were also detected in lacrimal and Meibomian glands of P0 (new born) mice. The reporter genes containing 1.5 and 2.6 kb 5’–Kera DNA fragments failed to express the ß–gal activity in any tissues of the transgenic mice. Use of intrastromal injection of the minigenes revealed that the 3.2 kb has highest ß–gal activity, the other reporter genes produced much less enzyme activity. Conclusions: The 3.2kb Kera promoter contains sufficient cis–regulatory elements for the reporter gene expression in cells expressing keratocan. The identification of ß–gal expression by the mesenchymal cells of lacrimal and Meibomian glands suggests that these cells are of neural crest origin.
Keywords: cornea: stroma and keratocytes • gene/expression • gene transfer/gene therapy