Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

Lin, Ya-Hui; Chang, Kung-Yao

HEK293T cells were plated in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Corning) in a 24-well plate one day before transfection. One hour before transfection, the medium was changed to Minimum Essential Medium ␣-Medium (␣-MEM, Gibco) containing 1% FBS. JetPrime TM transfection reagent (Polyplus) was used to transfect the reporter plasmids into 293T cells according to manufacturer's instruction. The medium was changed to fresh 1% FBS ␣-MEM containing final concentrations of 0.01, 0.1 or 1 mM of theophylline 4 h after transfection and transfected cells were cultured for another 18 h. A stable cell line harboring theophylline-responsive −1 PRF element embedded fluorescence reporter was established using a PiggyBac transposon system (25) . PBTPAF-theoOFF2-Switch1 was cotransfected into 293T cells with PB-RN and a helper plasmid PBCy43 (25) . Cells inserted with fluorescent reporter and rtTA were selected using a culture medium containing G418 (500 g/ml) and puromycin (10 g/ml). After 2 weeks of selection, surviving cells were treated with 1 g/ml tetracycline and reporter fluorescence was detected after 48 h of tetracycline treatment to identify a positive cell colony. This colony was named 293T-theo1.