EVALUATION OF ANTISENSE INHIBITION IN A B-CELL MODEL

An in vitro model has been developed in an attempt to optimise antisen se inhibition in B cells as a prelude to transgenic studies. The hypot heses tested were that i) the 3'-untranslated region would be an appro priate target for antisense inhibition; 2) the immunoglobulin heavy ch ain intronic enhancer could be used to enhance antisense inhibition vi a increased production of antisense transcripts; and 3) the mouse meta llothionein-1 promoter would allow induction of antisense inhibition i n B cells. Secreted IgM protein and mRNA were monitored following the stable transfection of a B cell line, HO-2.2, with a series of plasmid constructs containing antisense or sense target sequence DNA under th e control of either the mouse metallothionein-1 promoter or homologous tie same promoter as target sequence) immunoglobulin heavy chain prom oter. The 3'-untranslated region proved to be an appropriate target re sulting in 70% inhibition of IgM secretion. Compared with untransfecte d and sense controls, significant decreases in IgM secretion (and RNA levels) were: detected in clones transfected with antisense constructs utilising the mouse metallothionein-1 promoter and the immunoglobulin heavy chain intronic enhancer elements. These clones exhibited a furt her significant reduction in secreted IgM production upon zinc inducti on. Hybridisation studies demonstrated that decreased protein producti on was most likely attributable to reduction in RNA levels. In contras t, transfection with antisense constructs had no effect on membrane Ig M protein levels which not only confirmed the specificity of antisense action but meant that the: B cell remained sensitive to receptor liga tion. We conclude that reasonable antisense inhibition of gene product expression can be achieved in B cells by targeting the 3'-untranslate d region and using both an inducible promoter (mouse metallothionein-1 ) and the IgH enhancer to aid antisense RNA production.