Lr recombination reaction system

The generation of the lentivirus and adenovirus was performed as previously reported^{40 (link)}. Knockdown experiments for Pten were performed based on the lentivirus-mediated expression of the microRNA (miRNA) system using the BLOCK-iT Pol II miR RNAi Expression Vector Kit with EmGFP (Invitrogen). The sequences of DNA oligos for this purpose were designed using BLOCK-iT RNAi Designer provided by the manufacturer and hybridized oligos were inserted in the pcDNA6.2-GW/EmGFP-miR vector, followed by transfer into the pDONR vector by the BP reaction system (Invitrogen). To generate the lentivirus, the insert was recombined into the CSII-EF-RfA–IRES-puro vector (a kind gift from Dr. Hiroyuki Miyoshi (RIKEN BRC, Ibaraki, Japan) with the LR recombination reaction system (Invitrogen). The lentivirus was generated according to the manual provided by RIKEN BRC. UMR106 cells were infected with the lentivirus in the presence of 5 μg/mL polybrene (Sigma) and selected by puromycin (1 μg/mL). In the case of the generation of the adenovirus, the insert was recombined into the pAd-CMV-V5 vector (Invitrogen) using the LR recombination reaction system (Invitrogen). The adenovirus was generated using the ViraPower Adenovirus Expression System (Invitrogen) according to the manufacturer’s protocol.