20]In vivo analysis of signaling elements via targeted, inducible antisense RNA

Abstract

Summary Antisense RNA technology provides for selective suppression of proteins of interest and thus a new strategy with which to probe the emerging complexity of the various regulatory networks of signal transduction pathways. Considering sheer economics, the use of antisense DNA oligodeoxynucleotides is practical for studies requiring small-scale culture of cells, pilot studies seeking to test the antisense DNA strategy, and in systems in which single-cell assays can be performed (e.g., patch-clamping or histochemical analysis). Vector-driven antisense RNA expression, both constitutive and inducible, in cell culture allows for large-scale cell growth capacities enabling biochemical analyses. Expanding the antisense RNA approach to transgenic mice provides the means to generate unique animal models with which to explore the role of transmembrane signaling elements in complex biological processes in vivo. In our studies, suppression of Gsα with antisense DNA oligodeoxynucleotides provided exciting information concerning the role(s) of this G protein in adipogenesis ( 16 ). Similarly, the role of Giα2 in early mouse development has been addressed in F9 embryonic stem cells stably expressing antisense RNA ( 10 ). Finally, investigation of the role of Giα2 in adipose tissue and liver function as well as its role in whole-body metabolism, growth, and development have been made possible only through the hybrid PEPCK gene construct employed in our laboratory ( 11 ). Using a variety of antisense DNA/RNA-based approaches, investigators are now able to explore the roles of signaling elements at several distinctly different levels, selectively targeting the expression of a protein of interest in vitro or in tissues in vivo. Our knowledge of the role of transmembrane signaling elements in disease is growing rapidly. Our success with antisense DNA/RNA-based approaches in vitro and in vivo highlights the potential applications of this technology for use in gene therapy to treat pathological disorders. Expression of antisense RNA in a global set of tissues by retroviral infection or expression in a tissue-specific manner using selective promoters has implications not only for our basic understanding of how signal transduction pathways impinge on these complex events, but also for the development of new therapeutic agents with which to treat human disease.