Systematic analysis of antisense RNA inhibition of myosin II heavy-chain gene expression in Dictyostelium discoideum.

The powerful potential of antisense nucleotide inhibition of gene expression is presently being exploited in many biological systems. Although use of the technique is widespread, little is known about the variables that contribute to experimental success. We sought to define those variables that affect inhibition of myosin II heavy-chain (MIIHC) gene expression by stable nuclear-derived antisense RNAs in Dictyostelium discoideum. Different fragments of the MIIHC gene cloned in the antisense orientation into several transformation vectors were introduced into cells, and the accumulation of MIIHC protein and mRNA was examined. Inhibition of expression ranged from slight to virtually complete, and depended only on the specific gene fragment used to generate the antisense RNA. Fragments of the 3' end of the gene were the most effective and resulted in almost complete inhibition, whereas 5' fragments gave very little reduction. The severity of the morphological phenotypes associated with MIIHC depletion reflected the different levels of MIIHC in the transformants. Important implications for the design of antisense vectors suggested by these results are discussed.