All cell culture reagents were purchased from ThermoFisher Scientific unless otherwise noted. All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (D10). Cells were passaged by detachment with Trypsin-Ethylenediaminetetraacetic acid 0.25%.
Lentiviral vectors were produced by transfection of 250 000 HEK 293T cells in a 6-well with 500 ng pMD-VSV-G (Addgene #12259), 1750 ng PsPax2 (Addgene # 12260) and 1750 ng of the LLP or derivative vector template, using 6 μg of Fugene 6 (Promega). Media was changed the next day, and the supernatant collected over the next 72 h. The collected media was pelleted at 300 × g for 4 min, and the supernatant was passed through a 0.45 μm filter. HEK293T cells, A549 cells, NIH3T3 cells and HT-29 cells were incubated with various dilutions of lentiviral supernatant ranging from 100 μl to 4 ml, and transduced cultures were visually confirmed to have been transduced at MOI clearly less than one. Cells were then incubated with 10 μg/ml of Blasticidin. After a week of selection, cells were assessed with a BD FACS Aria II for fluorescence, and individual BFP+ cells were sorted into separate wells of 96-well plates. Cell clones that grew out were transferred into a 24-well plate and subsequently transferred to 6-well plates for analysis. Cells were induced to express off of the Tet-inducible promoter by adding doxycycline to a final concentration of 2 μg/ml (D10-dox). Cells were also maintained in D10-dox unless otherwise noted.
HEK 293T-based cells were recombined by transfecting 250 000 HEK 293T cells in a 6-well plate, with 1500 ng of pCAG-NLS-Bxb1, and 1500 ng of either individual or a mixture of attB recombination plasmids, incubated with 6 μg of Fugene 6 reagent in doxycycline-free media. Two or more days following transfection, the media was changed to D10-dox media, and cells were assessed for recombination at least 2 days after. For recombination of A549 cells, 150 000 cells plated in a 6-well with 2 ml of media were transfected with a mixture of 3.75 μl Lipofectamine 3000 diluted in 125 μl OPTI-MEM, and 5 μg DNA with 10 μl P3000 reagent diluted in 125 μl OPTI-MEM. For recombination of 3T3 cells, 150 000 cells plated in a 6-well with 2 ml of media were transfected with a mixture of 7.5 μl Lipofectamine 3000 diluted in 125 μl OPTI-MEM and 5 μg DNA with 10 μl P3000 reagent diluted in 125 μl OPTI-MEM. Recombined cells were positively selected by growing the cells for a week in D10-dox media supplemented with the indicated amounts of hygromycin, puromycin or blasticidin (Invivogen). Cells were split and media replenished every 3 days. Recombined cells were negatively selected with the addition of 10 nM AP1903 / Rimiducid (MedChemExpress). Cells were maintained in AP1903 for 2 days. Sodium Butyrate (Sigma) was dissolved as a 0.5 M stock solution in H2O.
Lentiviral vectors were produced by transfection of 250 000 HEK 293T cells in a 6-well with 500 ng pMD-VSV-G (Addgene #12259), 1750 ng PsPax2 (Addgene # 12260) and 1750 ng of the LLP or derivative vector template, using 6 μg of Fugene 6 (Promega). Media was changed the next day, and the supernatant collected over the next 72 h. The collected media was pelleted at 300 × g for 4 min, and the supernatant was passed through a 0.45 μm filter. HEK293T cells, A549 cells, NIH3T3 cells and HT-29 cells were incubated with various dilutions of lentiviral supernatant ranging from 100 μl to 4 ml, and transduced cultures were visually confirmed to have been transduced at MOI clearly less than one. Cells were then incubated with 10 μg/ml of Blasticidin. After a week of selection, cells were assessed with a BD FACS Aria II for fluorescence, and individual BFP+ cells were sorted into separate wells of 96-well plates. Cell clones that grew out were transferred into a 24-well plate and subsequently transferred to 6-well plates for analysis. Cells were induced to express off of the Tet-inducible promoter by adding doxycycline to a final concentration of 2 μg/ml (D10-dox). Cells were also maintained in D10-dox unless otherwise noted.
HEK 293T-based cells were recombined by transfecting 250 000 HEK 293T cells in a 6-well plate, with 1500 ng of pCAG-NLS-Bxb1, and 1500 ng of either individual or a mixture of attB recombination plasmids, incubated with 6 μg of Fugene 6 reagent in doxycycline-free media. Two or more days following transfection, the media was changed to D10-dox media, and cells were assessed for recombination at least 2 days after. For recombination of A549 cells, 150 000 cells plated in a 6-well with 2 ml of media were transfected with a mixture of 3.75 μl Lipofectamine 3000 diluted in 125 μl OPTI-MEM, and 5 μg DNA with 10 μl P3000 reagent diluted in 125 μl OPTI-MEM. For recombination of 3T3 cells, 150 000 cells plated in a 6-well with 2 ml of media were transfected with a mixture of 7.5 μl Lipofectamine 3000 diluted in 125 μl OPTI-MEM and 5 μg DNA with 10 μl P3000 reagent diluted in 125 μl OPTI-MEM. Recombined cells were positively selected by growing the cells for a week in D10-dox media supplemented with the indicated amounts of hygromycin, puromycin or blasticidin (Invivogen). Cells were split and media replenished every 3 days. Recombined cells were negatively selected with the addition of 10 nM AP1903 / Rimiducid (MedChemExpress). Cells were maintained in AP1903 for 2 days. Sodium Butyrate (Sigma) was dissolved as a 0.5 M stock solution in H2O.
