TRANSIENT EXPRESSION ASSAY FOR ANTISENSE RNAS USING EPISOMAL REPLICATION OF PLASMIDS - EFFECTIVE REDUCTION OF RETINOBLASTOMA GENE (RB-1) PRODUCT BY ITS ANTISENSE RNA COMPLEMENTARY TO 3'-UNTRANSLATED REGION

We have developed a transient expression assay for selection of effect ive antisense RNAs using episomal replication of plasmids in COS-7 cel ls, an African green monkey kidney-derived cell line expressing SV40 l arge T antigen. The transient expression assay was enabled by a liposo me-Mediated DNA transfection method, by which about 70% of the cells w ere reproducibly transfected with exogenous DNAs. Plasmids expressing antisense RNAs for the retinoblastoma gene (Rb-1) mRNA and harboring S V40 ori were constructed and introduced into COS-7 cells to examine th eir inhibitory effect on the accumulation of endogenous Rb protein (pR b). Only the antisense RNA complementary to the 3'-untranslated region (UTR) in Rb-1 mRNA was expressed stably at high levels for 3 days aft er the transfection. This antisense RNA reduced by 73% the content of endogenous pRb 70 h after transfection. A similar inhibition was detec ted in mouse mammary carcinoma cells (FM3A) that were stably transfect ed with the antisense RNA expressing vector directed to 3'UTR. In cont rast, no obvious change in pRb was observed with antisense RNAs comple mentary to the coding region of Rb-1 mRNA. The cellular content of the se antisense RNAs was lowered by degradation; thus these RNAs did not affect the levels of pRb in COS-7 and FM3A cells. These results, taken together, suggest that the expression levels and the stability of ant isense RNAs are involved in their repressive activity, and our transie nt expression assay provides a rapid and easy system for evaluation of ectopic antisense RNA activity in COS-7 cells.