Purpose: The authors used and validated a recently developed method, mRNA differential display, to detect and clone genes that are differentially expressed in healing compared to stationary corneal epithelium.
Methods: RNAs from unwounded and 18-hour postwound corneal epithelia were isolated and subjected to mRNA differential display analysis. The generated cDNAs were used as probes in Northern blot analysis and in situ hybridization to confirm their differential expression and to clone longer or full-length cDNAs from a healing corneal epithelial cDNA library.
Results: Changes in the pattern of gene expression in healing epithelium, compared with that in stationary cells, were noted. To date, 15 combinations of 5'- and 3'- primers were used with approximately 1500 mRNA species screened. Differential expression of nine mRNA species were observed. These included four known proteins. They are nonmuscle tropomyosin TM-1, cytokeratin K14, small GTP binding protein rab 11, and amyloid beta-A4 precursor-like protein-2. One is a sequence with homology to type II cytokeratin, and four represent genes with sequences that are unreported. The differential expression of five of these genes was confirmed by Northern blot analysis, in situ hybridization, or both.
Conclusion: mRNA differential display provides a unique and powerful experimental system to study differential gene expression in wound healing and cell migration. Using this system, differential expression of nine genes was observed. Detection of genes differentially expressed in healing epithelium may prompt studies that will define the specific role of each of the proteins in wound healing.