Application of a dicistronic mRNA expression vector to antisense RNA expression in mammalian cells

One important role of antisense RNA expression in mammalian cell biology is to partially suppress the functions of target genes of which the functions are unknown but which are indispensable for host cell growth. Therefore, we applied a dicistronic mRNA expression vector to express antisense p53 RNA in mouse fibrosarcoma MethA cells. We also established several clones in which the p53 gene functions were partially suppressed by the introduced antisense RNA. Compared with the control MethA clones, the contents of p53 protein in those expressing the antisense RNA were about halved, and their growth rates were remarkably decreased. These results proved that an antisense RNA in a dicistronic mRNA construct decreases the cellular content of a targeted gene product and that clones harboring the dicistronic mRNA construct can be established in which expression levels of the introduced antisense RNA are controlled by the stringency of drug selection.