Use of Triplex-Forming Oligonucleotides and Adenoviral Constructs for Studying the Regulation of Gene Expression

Short synthetic homopyrimidine- or homopurine-rich oligonucleotides can form sequence-specific triplexes with corresponding homopurine–homopyrimidine sites on duplex DNA and block transcription of a target gene in vitro. Such triplex-forming oligonucleotides (TFOs) can be rationally designed to target homopurine/homopyrimidine sequences that are often found in eukaryotic genes and thus used to modulate the expression of these genes. The antigene strategy using TFOs has been successfully applied to a number of genes in vitro. In this article we describe methods used in applying this antigene approach to the rat aquaporin 5 (rAQP5) gene. We specifically focus on the selection of TFOs based on the sequence of the target gene and on a novel method employing adenoviruses for delivery of TFOs to cells in vitro.