Protransduzin a

—Jurkat cells were incubated in 500 μl lentivirus and 50 μg/μl Protransduzin-A (Immundiagnostik) for 5 min at RT. The Jurkat cells were transferred to fresh media after 4 h, and were selected with 1 μg/μl puromycin (AppliChem). Transduction efficiency was determined by FACSCanto II. The knockdown was verified by immunoblotting.

The murine HPSE transgene was co-expressed with an eGFP-Zeocin marker from a polycistronic message driven by the minimal EF1a promoter utilizing a self-cleaving P2A peptide. Lentiviral vectors were packaged in HEK 293 T cells as previously described. Briefly, 10 µg of transfer plasmid was cotransfected with helper plasmids (10 µg of pCMV-Pol/Gag, 4 µg of pCMV-Rev, and 4 µg of pCMV-VSVG) into HEK 293 T cells at 80% confluence using Fugene 6 (Promega). Viral supernatant was harvested 48 h post transfection, concentrated by ultracentrifugation, and stored at −80 °C until use. Viral titers were determined by transduction of HT1080 cells and analyzed for GFP expression by FACS. CTLL-2 cells were transduced with viral supernatants at an MOI of 10. Protransduzin-A (Immundiagnostik AG) was added according to the manufacturer’s instructions and transduction reactions were spinoculated at 1000 g for 90 min at 32 °C in sealed tubes. An equal amount of fresh medium was added and cells were incubated in the presence of transduction complex for an additional 4 h at 37 °C. The transduction medium was replaced with fresh medium and cells were cultured for 48 h before starting selection with 200 µg/ml Zeocin. After selection for 7 days >99 % of cells were GFP positive by flow cytometry. Full vector sequences are available upon request.