Discordance between gene regulation in vitro and in vivo.

Mam m alian gene expression can be regu­ lated by cell type, developm ental stage, and num erous external stimuli. Regulation is b rough t about by a complex interaction of cisacting DNA sequences and trans acting factors. Considerable effort has been expended to iden­ tify and characterize regulatory DNA sequences and to understand the basic m echanism s by which they exert their actions. Traditionally, the transcriptional activities o f putative prom oters and regulatory sequences have been m easured by transfections into cultured cells o f various DNA constructs consisting o f these sequences fused to a repo rte r gene and subsequent quan­ tita tion of repo rte r gene expression. This ap ­ proach has provided a great deal o f inform a­ tion concerning the sequences m ediating the expression of genes in various cell types in re ­ sponse to physiologic perturbations such as horm ones —and, to a lesser extent, during spe­ cific developm ental stages, such as the fusion o f myoblasts to form myotubes. Given the complexity o f biological processes in vivo, however, a question arises w hether reg­ ulatory elem ents identified in cultured cells accurately explain the regulation o f gene ex­ pression in vivo. This becomes particularly im ­ po rtan t in considering the use o f im m ortalized cell lines or prim ary cultures, whose patterns o f gene expression often differ significantly from those o f the intact tissue. O ne way to evaluate the relevance o f in vitro transfection studies to gene regulation in vivo is to create transgenic anim als using constructs that have also been tested in cultured cells. This approach requires the generation o f several in ­ dependent mouse lines for each construct under study, an expensive and time-consuming u n d e r­ taking com pared with transfections into cul­ tu red cells. O ne alternative to transgenic mice that may approxim ate in vivo gene regulation m ore closely than cell culture transfections is the injection of DNA sequences linked to re ­ porters into cardiac and skeletal muscle tissue in vivo (Kitsis et al., 1991). However, this gene transfer technique appears to be lim ited to stri­ ated muscle cells (Wolff et ah, 1991). Regardless o f w hether gene regulation is studied in vitro or in vivo, the state of the for­ eign DNA may influence the regulation of its expression. W ith transient transfections into cul­ tu red cells o r injections of DNA into muscle in vivo (Wolff, et ah, 1990), the construct being tested exists in an episom al state. The geom et­ ric configuration of the regulatory sequences in the construct may differ m arkedly from its natural configuration in the endogenous locus. This, in turn , may influence regulation o f the expression of the construct. In addition, an episome will not be subject to regulatory in fo r­ m ation that may reside in the chrom atin con­ figuration. O n the o ther hand, with stably transfected cells and transgenic animals, the con­ struct undergoes integration into random chro­ m osom al sites whose chrom atin configuration may differ from that o f the endogenous locus. In fact, the im portance of long-range chrom o­ some effects is supported by the existence of locus control regions (LCR), such as those seen in the p globin gene com plex (see Grosveld, et