Adeno gfp

Adeno-GFP (Vector BioLabs, Cat. No. 1060) or Adeno-LifeAct-GFP or -RFP (Ibidi, Cat. No. 60121 and 60122) were added after organoid isolation, prior to suspension in ECM. Isolated organoids were centrifuged in an Eppendorf tube at 520 g for 10 min. The supernatant was removed and organoids were resuspended in 100 μl DMEM-F12 (Gibco). Adenovirus was added at 5000-10,000 plaque-forming units per organoid to achieve gene expression in ~50 percent of cells. Organoids were incubated with virus for 1 h at 37°C and then washed twice with DMEM-F12 and suspended in ECM for plating. Lentiviruses encoding Raf1-(RBD)-GFP (Bondeva et al., 2002 (link); Sasaki et al., 2004 (link); Taylor and Shalloway, 1996 (link)) and PH-Akt-GFP (Addgene #51465) (Varnai and Balla, 1998 (link)) mixed at 100,000 PFUs with 3 μL of Viromag (OZ Bioscience, #RL400200) into 50 μL of DMEM-F12 and incubated for 30 min at RT. Organoids were plated at 1,500 orgs per well in a 96-well non-adherent plate (Thermo, #174907) and allowed to gravity settle for 30 min. Lentivirus was then mixed with organoids and incubated on top of a magnetic plate (OZ Bioscience, #MF10000) for 90 min and then incubated overnight at 37°C. Organoids are then washed twice with DMEM-F12, suspended in ECM, and plated.

Prior to embedding in collagen I, freshly isolated MMTV-PyMT organoids were infected with adeno-Cre (Vector BioLabs, 1045) at an MOI of ~10⁷ per 800 organoids. Infection was carried out in suspension, in 50 μL of DMEM-F12 overnight at 37° C. This typically yielded a recombination efficiency of 75–85%. Similar methods were followed for E-cad knockout in C3(1)-Tag tumor organoids. In this case, control organoids were treated with adeno-GFP (Vector BioLabs, 1060) using identical conditions.