Stable transfection. The GPC1 antisense construct was prepared by RT-PCR amplification of human placenta cDNA, as described previously (11). Stable transfection of GPC1-AS-1751 into PANC-1 cells was performed by using the lipofectamine method (12), and single clones were isolated after 3-4 weeks. After expansion, cells from each individual clone were screened for expression of GPC1 sense and antisense mRNA by northern blot analysis. Parental PANC-1 cells also were transfected with an empty expression vector carrying the neomycin-resistance gene as a control. Positive clones were routinely grown in selection medium.