Specific gene suppression by engineered ribozymes in monkey cells.

Abstract

Short catalytic RNAs possessing specific endoribonuclease activity (ribozymes) have recently been designed that can potentially shear any chosen target RNA in trans at a specific site. Here, engineered ribozymes targeted against chloramphenicol acetyltransferase (CAT), derived from Tn9, have been cloned into a mammalian expression vector and tested in transient transfection experiments for their effects on CAT expression in monkey (COS1) cells. The ribozymes contained the catalytic domain of the satellite RNA from tobacco ringspot virus and were targeted to three sites in the CAT mRNA by flanking antisense sequences. These ribozymes, which were previously shown to accurately cleave CAT message in vitro, were cloned into a replicating plasmid vector under the control of the highly active simian virus 40 early promoter. The ribozyme gene sequence was incorporated into the 3' untranslated region of the gene for firefly luciferase as it was ineffective when expressed as a short RNA. Each ribozyme construction gave a similar level of suppression of CAT activity when the target was transcribed from the herpes virus thymidine kinase promoter. One of the three (ribozyme 2) was chosen for further study and tested after it had been modified by the addition of extra flanking bases. The reporter gene for luciferase was used to monitor ribozyme level and to function as a specificity control, and the human growth hormone gene was cotransfected as an independent reporter for specificity of the ribozyme against the intended target CAT. At high (approximately 1000-fold) molar excess this ribozyme was demonstrated to consistently and specifically suppress CAT expression (up to approximately 60%) in COS1 cells relative both to a plasmid clone with the ribozyme inserted in the reversed (inactive) orientation and to a control corresponding to the relevant 26-nucleotide antisense segment of CAT.