The identification of novel wound-healing genes through differential display.
Effective methods to identify novel genes in complicated dynamic tissue processes are needed in molecular biology research. Traditional techniques primarily target known genes and are inefficient in the pursuit of unknown genes. Here we describe the use of a modified differential display polymerase chain reaction (DD-PCR) protocol for the identification of genes differentially expressed in wound healing. Full-thickness dorsal wounds were made on 35 adult rats, followed by wound harvest at 12 hours, 24 hours, 3 days, 5 days, 7 days, 10 days, and 14 days after injury. Modified DD-PCR was performed and gene fragments displaying definite changes during wound healing were cloned and sequenced. Gene fragments from DD-PCR were compared with available gene bank database sequences. Specific primer PCR was used to confirm DD-PCR expression patterns. As a result, over 1000 gene fragments were amplified by DD-PCR, 35 of which demonstrated distinct differences during repair. Cloning and sequencing of 13 of these gene fragments revealed that some were homologous to several characterized genes with previously unsuspected roles in repair, whereas others were completely novel genes with no known function. Specific primer PCR further confirmed expression of six of these 13 gene fragments. Only one of the 13 cloned fragments, later identified as interleukin-1beta, had well-recognized associations with tissue injury. Other fragments corresponded to various genes involved in cellular processes such as differentiation, proliferation, exocytosis, and myofibril assembly. No prior studies have linked them to wound healing. We have demonstrated that modified DD-PCR can be used to effectively identify novel genes differentially expressed during repair. Because DD-PCR allows for the simultaneous amplification of multiple arbitrary transcripts, it is a powerful genetic screening tool for complicated dynamic tissue processes, particularly when multiple, limited-sized samples are involved.