Construction and Expression of Oligonucleotide-Based Antisense Cassettes: FIGURE 1.

INTRODUCTION This protocol describes simple and robust methods for the construction and cloning of expression constructs that can be used to deliver antisense effectors, exemplified by the hammerhead ribozyme and short hairpin RNA (shRNA), into cultured cell lines. The protocol also describes the construction of reporter vectors to be used for target validation. Due to the variable efficacy of the antisense effectors, it is advisable to design multiple constructs targeting different sites. Once the different constructs have been generated, their relative efficacy can be readily determined through reporter cotransfection experiments, in which a stretch of cDNA encompassing all target sites is cloned directionally into the 3′ UTR (untranslated region) of a reporter (psiCheck-2; Promega). Successful cleavage of the target site results in degradation of reporter mRNA, with concomitant decrease in translated product, which is detected by a luminescence-based assay system.