Lentix titration kit

Pancreatic organoids were infected with a two-vector lentivirus-based Tet-on system (Clontech). Coding sequences for HA-tagged WT and 6S-A Ngn3, fused with GFP-2A cleavage peptide sequence at their N-terminus, were cloned into the pLVX-TRE3G vector (Clontech). Viruses were generated in HEK293T cells, titrated with the LentiX titration kit (Clontech) and used at multiplicity of infection of 10 for the transgene, or 20 for the transctivator Tet3G. Organoids were dissociated to small clusters by TrypLE (Gibco) treatment for 10 min at 37⁰C. Dissociated organoids were incubated with the viruses and 8μg/ml polybrene (Sigma) in expansion media supplemented with 10μM ROCKI (Sigma) and spun for 1h at 300xG at room temperature. After spinoculation, infected organoids were incubated in a cell culture incubator at 37⁰C for 5-6 hours before plating in matrigel with fresh media supplemented with ROCKI.

Lentiviral particles were produced in HEK293T cells as described in (Azzarelli et al., 2018a (link)). Briefly, HEK293T cells were transfected with third generation packaging vectors (vectors containing VSVG, gag-pol, rev and tat in a ratio 7:1:1.1) and with pBob-H2B-GFP vector (kind gift from Professor Rick Livesey), using Calcium Phosphate method (Promega ProFection^® kit, E1200). The morning after transfection, the medium was refreshed and then collected for viral production 36 h later. To concentrate the viral particles, the medium was mixed with the concentrator (Lenti-X™ Concentrator, Clontech, 63123) overnight at 4°C and then spun for 45 min at 2000× g at 4°C. After removing the supernatant, the pellet containing viral particles was resuspended in 200–300 µl of medium and stored in 20 µl aliquotes at −80°C. Viral titer was quantified using Titration kit (Lenti-X™ Titration kit, Clontech, 631235); the viral particles needed have been calculated using the formula: viral particle=[(total number of cells×MOI-multiplicity of infection)/titer)]×1000. The MOI used in our experiments is 10.