Piggybac transposase expression vector

1 × 10⁶HMOX1-targeted hiPSCs (clone H7-03) were nucleofected with 2 µg Excision Only PiggyBac™ Transposase Expression Vector (SBI) then plated onto 10 cm dishes for colony picking. 10 days after nucleofection, individual colonies were isolated and transferred into organ dishes for separate propagation. DNA analysis and puromycin-sensitivity testing of the cells were performed after three passages.

The following vectors were used: lentiCRISPRv2 (a gift from Feng Zhang, Addgene plasmid # 52961) and pUC19 (gift from Joachim Messing, Addgene plasmid # 50005). The pMAX-GFP (VDF-1012) was supplied by the Amaxa nucleofection kit. The PiggyBac hybase plasmid is generously provided by Prof. Jacob Giehm Mikkelsen from the Department of Biomedicine, Aarhus University (http://www.giehmlab.dk). Alternatively, the PiggyBac transposase expression vector can be purchased from SBI system Biosciences (Cat. # PB210PA-1).

We established HEK293 cell lines that stably express N-terminal 3xFLAG-tagged human CDKN1C cDNA (WT or Arg316Gln) in the presence of doxycycline. The doxycycline-inducible piggyBac backbone vector has been described previously [42 ]. We introduced the human CDKN1C cDNA sequence (WT or Arg316Gln) into the backbone vector with the Gibson assembly technique. HEK293 cells were co-transfected with each piggyBac vector and the piggyBac transposase expression vector (System Biosciences, Mountain View, CA, USA) using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Stable cells were established according to the manufacturer’s protocol.
Whole cell lysates were prepared from inducible stable cells maintained with or without 1 μg/mL doxycycline for 48 h. We performed Western blotting with anti-FLAG M2 antibody (Sigma-Aldrich, St Louis, MO, USA) and anti-tubulin antibody (Abcam, Cambridge, UK) as primary antibodies.