Antisense-induced ribosomal frameshifting

Henderson, Clark M.; Anderson, Christine B.; Howard, Michael T.

Plasmid p2lucAZ1PKdel was co-transfected into CV-1 cells with varying concentrations of AZ1B 2 0 -O-Methyl antisense oligonucleotides under the following conditions. CV-1 cells (1.5 · 10 4 ) in 50 ml of DMEM + 5% fetal bovine serum were added to wells (1/2 area 96-well tissue culture treated plates) containing 25 ng of DNA, varying amounts of AZ1B antisense oligonucleotides and 0.4 ml Lipofectamine 2000 (Invitrogen) in 25 ml of Optimem. Cells were incubated at 37 C (5% CO 2 ) for 20 h. Media were then removed from the cells and the transfected cells were lysed in 12.5 ml lysis buffer and luciferase activity determined by measuring light emission following injection of 25 ml of luminescence reagent (Promega). Percent frameshifting was calculated by comparing firefly/Renilla luciferase ratios of experimental constructs with those of control constructs: (firefly experimental RLUs/Renilla experimental RLUs)/(firefly control RLUs/Renilla control RLUs) · 100.