Padvantage plasmid

Human embryonic kidney 293T (HEK293T) cells grown in SensiCell™ medium supplemented with 10% foetal calf serum (FCS) were plated into 6-well plates and left overnight to achieve 70% confluency. Cells were then washed twice with Dulbecco’s modified Eagle medium and transfected using Xfect™ (Clontech) in serum free medium for 4 h, with a total of 3 µg DNA consisting of the TRE.ChABC and PGK.GARrtTA plasmids in a ratio of 1:4 and 0.3 µg pAdVAntage™ plasmid (Promega). pAdVAntage™ was added to enhance expression of the ChABC gene (Muir et al., 2010 (link)). Following transfection, medium was removed and replaced with doxycycline-free medium containing 10% FCS (Clontech). Doxycycline was then added at 1, 10 and 100 ng/ml. Medium and cells were harvested after 48 h and enzyme levels in the medium measured by enzymatic colorimetric assay (Morgan Elson reaction), n = 5, 3, 3 and 6 for 0, 1, 10 and 100 ng/ml, respectively, where n = individual well normalized to the maximum activity in each plate.

Expifectamine transfection kit, polyacrylamide gels, biotin-NHS, Zeba columns, and AcTEV protease were from ThermoFisher. Growth media was from ThermoFisher or BD Difco. pAdvantage plasmid and CellTiterGlo were from Promega. MabSelect, HisTrap, Q Sepharose, and Superdex columns were from GE Healthcare. TSKGel column was from TOSOH. PNGase F was from NEB. Triglycine peptides were from Anaspec. Monomethyl auristatin F (MMAF) molecules were from Levena Biopharma. Modified oligonucleotides were from Oligo Factory or Biosynthesis.

Approximately 1 × 10⁷ HEK293T cells were transfected with 1.7 µg pAdvantage plasmid (Promega), 2.6 µg VSV-G expression vector, 4 µg gag-pol expression vector and 6.6 µg CreERT2 expression vector, using TurboFectin transfection reagent (OriGene). Medium was changed 24 hours after transfection. Viral supernatant was collected 48 and 72 hours after transfection, and used to transduce the METTL16-DECAI cells. Since the Cre-ERT2 retrovirus also encoded a PGK-PuroR cassette, cells containing Cre could be enriched for by selecting with 0.3 µg/ml puromycin.