We have been developing triple ribozyme (TRz) constructs which consist of two cis-acting ribozymes flanking an internal trans-acting ribozyme, which is targeted to a cellular RNA. Actions of the two cis-acting ribozymes efficiently liberate the internal ribozyme with minimal nonspecific flanking sequences. The liberated internal targeted ribozyme shows substantially greater catalytic activity than TRz preparations, constructs which cannot undergo self-liberation or than single ribozymes with flanking vector sequences. Here we construct a TRz which was targeted to retinoblastoma gene (Rb) mRNA, which cleaved Rb target RNA in vitro as expected. A number of tetracycline-regulatable clones stably transfected with the Rb-targeted TRz were developed and analyzed. The internal targeted ribozymes were efficiently liberated in vivo and the stably transfected clones showed varied reductions in Rb mRNA, which were contingent upon ribozyme expression and catalytic activity. The two clones showing major reductions in Rb mRNA (and pRb) levels (> 70% reduction) showed abnormal morphology, loss of contact inhibition and the ability to grow in soft agar, as well as altered compartmentation of repetitive B2 transcripts, a phenomenon previously associated with immortalization and/or transformation. TRz constructs coupled with tissue-specific promoters should allow development of in vivo models in which Rb function is markedly reduced in a tissue-specific manner.
All Science Journal Classification (ASJC) codes
- Cancer Research