Figure 3

MDR1 antisense-mediated down-regulation of P-glycoprotein (P-gp). (A) RT-PCR reactions on mRNA from wild-type Caco-2 cells and Caco-2 subclone R34, generated by transfection with MDR1 antisense gene cloned into the pEUK-c1 vector. A specific band (arrow) of antisense MDR1 mRNA was found in clone R34. Parental Caco-2 cells did not show this amplification product. The positive control PCR on the pEUK-c1-RDM vector gives a product 965 bases longer, as the vector contains an intron. (LM) ladder marker, representing different molecular lengths). FACS analysis of (B) wild-type Caco-2 cells and (C) MDR1-antisense transfectants R34 for the expression of P-glycoprotein. The cells were incubated with an non-specific mouse-antibody and FITC-labeled anti-mouse-antibody (isotype control), and specifically labeled with the monoclonal antibody MRK16 and FITC-labeled anti-mouse-antibody (P-gp expression). Isotype control analysis did not reveal significant numbers of cells that would be considered P-gp positive. Compared to non-transfected cells (79.4%), the P-glycoprotein expression in clone R34 was significantly reduced (41.8%) (Pictures taken from [73]).