IDENTIFICATION OF CANDIDATE GENES FOR DRUG DISCOVERY BY DIFFERENTIAL DISPLAY

Regulation of gene expression can specify cellular fate, define respon ses to stimuli, and contribute to complex microenvironments present in tissues. Identification of differentially expressed genes in experime ntal paradigms can help elucidate underlying biochemical pathways and thus reveal potential therapeutic targets. The technique of differenti al display uses arbitrarily primed PCR to sample complex cDNA populati ons of interest; amplified portions of messenger RNAs are analyzed by denaturing gel electrophoresis and those which are differentially repr esented can be directly visualized and cloned. PCR-based techniques fo r analysis of gene expression are reliable and extremely sensitive. in comparison to traditional methods, such as subtractive hybridization, differential display allows for many samples to be compared in parall el, and the requirement for starting material is low. There are a plet hora of examples in the literature of how differentially expressed gen es can be rapidly identified in experimental paradigms ranging from ce lls treated in culture to whole organs of treated animals. The challen ge for the researcher is then defining candidate genes for drug discov ery from an initial screen based only on differential expression patte rns. Careful experimental design and execution are critical for optima l use of such methodologies to fill a gene discovery pipeline. In this article, the merits and potential pitfalls of differential display an d related PCR-based techniques are discussed. Current protocols are re viewed and innovations pertaining to high-throughput applications are noted. (C) 1997 Wiley-Liss, Inc.