Lentivirus DNA clones of control and Piwil1 shRNAs were purchased from Sigma-Aldrich. Flag-Piwil1 was generated by PCR from cDNA and cloned into the pCDH-MCS-T2A-Puro-MSCV lentiviral vector (System Biosciences), CCND2, MCL1 and OLIG2 were generated by PCR from cDNA and cloned into pCDH-EF1α-MCS-IRES-neo lentiviral vector (System Biosciences).
Lentivirus were produced in 293T cells. Briefly, lentivirus plasmids were transfected with packaging plasmid psPAX2 and envelope plasmid VSV-G (at a ratio of 1:1:1) into 293T cells. After 12 hours, the media were changed to neural basal media and incubated for another 48 hours. The collected mediums were passed through 0.45 μm filters and stored at −80°C.
Lentivirus were produced in 293T cells. Briefly, lentivirus plasmids were transfected with packaging plasmid psPAX2 and envelope plasmid VSV-G (at a ratio of 1:1:1) into 293T cells. After 12 hours, the media were changed to neural basal media and incubated for another 48 hours. The collected mediums were passed through 0.45 μm filters and stored at −80°C.