Effects of different mRNA 3'-untranslated regions on hammerhead ribozyme efficiency in vivo

Abstract

880-01The efficiency of ribozyme function depends on the promoter as well as the stability and localization of the transcript. Thus expression cassette determine the functional activity of ribozymes by controlling their intracellular colocalization. We have studied hammerhead ribozyme function suppressing Hepatitis B Virus X gene. Especially, we have designed the expression cassette including β- and γ-actin 3-UTR(untranslated region). Actin 3-UTR sequences are responsible for localization of mRNA and we acquired these sequences by PCR with HeLa cDNA library. pRc/RSV was used as mammalian cell expression vector and several plasmids containing riobzyme and target flanked by each 3-UTR were constructed. After each kind of ribozyme and target were paired off, these vectors were transiently transfected into HeLa cell and ribozyme activities were detected by β-galactosidase assay. When the cells were transfected with vectors including 3-UTR in either target or ribozyme, the activity of ribozyme was reduced. But the cells were tranfected with vectors including the same kind of 3-UTR in both target and ribozyme, the activity of ribozyme was slightly enhanced.