Tet on inducible gene expression system

HeLa Kyoto, HeLa Tet On, HPNE, LN9 and EUFA423 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% FBS, 100 U/ml penicillin and streptomycin. Cells were maintained at 37 °C with 5% CO₂. The human BRCA2-deficient fibroblast cell line, EUFA423, and human fibroblast cell line obtained from clinically healthy individuals LN9 was a kind gift from VU University Medical Center (Amsterdam, The Netherlands) (53 (link)). The reconstituted cell line (EUFA423 + BRCA2) was generated by introducing FLAG tagged full-length BRCA2 cDNA construct into the human BRCA2-deficient fibroblast cell line EUFA423 cells as described previously (54 (link)). BRC4 expressing cell line (HeLa Tet On clone 4.23) was generated using Tet-On Inducible Gene Expression System (Clontech) as described previously (55 (link)); this cell line expresses BRC4 repeat (BRCA2 residues 1481–1553), C-terminally tagged with myc epitope and 3xNLS (nuclear localization signal). hTERT-HPNE human ductal pancreatic cell line was purchased from Lgcstandards. Cells were plated in 8-well, glass-bottom (1.5 thickness) ibidi μ-slides (ibidi, 80827).

HeLa cells were transfected with a Tet-On inducible gene expression system (Clontech) and set to target the I-Sce1 gene according to the Tet-On system manual. After generation of a stable Tet-On cell line, the cells were transfected with the GFP reporter construct for NHEJ provided by the Gorbunova lab [40 (link)]. These cells were grown under selective pressure with Geneticin (G418) to generate the stable iHN20.22 cell line containing the reporter construct and a tetracycline inducible I-Sce1 gene.