Generation and Characterization of CIZ1-null Mouse Primary Cells

CIZ1-null mice were generated as described [14 (link)] from C57BL/6 ES clone IST13830B6 (TIGM) harbouring a neomycin resistance gene trap inserted into intron 1. Murine primary embryonic fibroblasts (PEFs) were derived from individual embryos at days 13–14 of gestation and murine tail-tip fibroblasts (TTFs) at 3–4 weeks (Additional file 7: Table S2). Both were maintained in Dulbecco's Modified Eagle Medium (DMEM), GlutaMAX, high glucose (4.5 g/l), and low glucose (1 g/l) (Gibco), respectively, and grown on Nunc™ Cell Culture Dishes (ThermoFisher Scientific, 150,350) with 0.13–0.16 mm thick glass coverslips. Media was supplemented with 10% FBS (PAA) and 1% Pen/Strep/Glutamine (PSG, Gibco) referred to here as high-serum media. Cells were maintained in a rapidly cycling state at 37 °C with 5% CO₂ and split to avoid cell contact. All cell populations were used at passages 2–4. For inducible cells harbouring transactivator and responder transgenes, addition of doxycycline (dox) to media (5–10 μg/ml) was used to induce GFP-CIZ1 over 48 h.