Application of differential display to immunological research

The majority of immunological processes are mediated by cell-to-cell contac t or receptor-ligand interactions that transmit intracellular signals and a ffect the regulation of transcription in the nucleus. As a consequence, pre cursor cells develop into their respective lineages and cells differentiate further during an immune response. In order to study changes in normal cel ls or even cells that have been isolated from diseased tissue, a number of approaches have been developed. One such method, differential display (DDRT -PCR), is a versatile technique for the analysis of gene expression that is based on RT-PCR and denaturing polyacrylamide gel electrophoresis. This te chnique is applicable to multiple samples of clonal or purified cell popula tions as well as to complex tissues and can be used to provide mRNA fingerp rints. However. the main purpose of DDRT-PCR is to isolate differentially r egulated genes in biological systems. The method is carried out without pri or hypothesis as to which genes should be examined and so increases the pos sibility of identifying completely novel and unexpected changes in transcri ption. A major drawback has been the isolation of false positive clones and the need to confirm the results of analysis by another method. This makes DDRT-PCR labour intensive. A number of strategies have been recommended to reduce these problems, including reverse-northern analysis as a confirmator y step for screening putative differentials. In order to reduce the number of gel fingerprints that would be required to cover all the mRNAs in a cell , several focused approaches have been suggested. These include targeted di fferential display for the isolation of multigene families that have conser ved protein domains or gene signatures and subtractive differential display whereby one population is subtracted from the other prior to screening. Th e purpose of this review is to provide some guidance to the immunologist wh o might wish to apply DDRT-PCR in their research. A number of examples wher e DDRT-PCR has been used successfully in immunological research are include d. (C) 2001 Elsevier Science B.V. All rights reserved.