Lentivirus generation and p53 knockdown in 253G1 feeder-free iPS cells

p53 specific sequence and control shRNAs were obtained from Addgene (Cambridge, MA, USA). Lentiviral particles were produced using the second-generation packaging system obtained from Addgene as reported previously⁴¹. In short, 10 μg of p53 or control shRNA plasmid, 7.5 μgs of packaging plasmid (psPAX2 plasmid) and 2.5 μg of envelope plasmid (pMD2.G plasmid) were transfected in HEK293t cells using FUGENE 6 (Promega, Fitchburg, WI, USA) transfection reagent according to manufacturer’s instructions. Medium was replaced 24 hrs following transfection. Two days later, lentivirus containing medium was collected and concentrated using Lenti-x concentrator (Takara Bio, Mountain View, CA, USA). Lentivirus particles were re-suspended in OPTI-MEM (Life Technologies), aliquoted and stored in −80 °C for later use. 253G1 human iPS cells were transduced with lentiviral particles using 6 μg/ml polybrene (San Cruz Biotech). When confluent, transduced cells were passaged and expanded. Successfully transduced cells were selected using 2 μg/ml puromycin and were used for experiments.