23] Antisense DNA/RNA-based strategies to analysis of signal transduction via G proteins

Abstract

Summary The ability to selectively suppress the expression of specific signaling elements has provided a new strategy with which to probe the complex regulatory networks of signal transduction pathways. From an economical perspective, the use of antisense DNA oligodeoxynucleotides is practical for studies which require relatively small-scale culture of cells, for pilot studies seeking to test the antisense DNA strategy, and for cell systems amenable to single-cell assays (i.e., 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 animals provides the means to generate unique mouse models with which to explore the role of transmembrane signaling elements in complex biological processes in vivo. In our studies, the use of antisense oligodeoxynucleotides for suppression of Gsα or for suppression of specific protein kinases provided powerful insights into the roles of these proteins in differentiation and receptor desensitization, respectively. Similarly the role of Giα2 in stem cell differentiation and the role of PKC in receptor desensitization have been addressed in cells stably expressing antisense RNA. 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 has been made possible only through the hybrid PEPCK gene construct employed in our laboratory. Using a panel of different antisense DNA/RNA-based approaches, one can explore the roles of signaling elements at several distinctly different levels by selectively suppressing either a single target or a family of targets in cells 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. The delivery of anti-sense DNA oligodeoxynucleotides or retroviruses harboring antisense RNA sequences to tissues as well as the ability to express antisense RNA in a narrowly defined and specific set of tissues has great implications not only for our basic understanding of how signal transduction pathways impinge on these complex events but also for the treatment of human disease.