Cell line targeting

The RMCE acceptor ES cell line (CGL1.10E; 129S7 genetic background) was kindly provided by Haydn Prosser (Sanger Institute, Hinxton, UK). ES cells were cultivated on mitomycin C treated SNL Feeder Cells (kindly provided by Haydn Prosser). The targeting protocol was adapted from a previous published method for RMCE (Prosser et al. 2008). Briefly, 10.5 million trypsinized cells were mixed with 10 μg of CsCl purified BAC and 25 μg of pCAGGS-Cre (kindly provided by Olga Ermakova, CNR) in a 0.4 μm cuvette (Bio-Rad) and electroporated (230 V, 500 μF). Cells were collected, centrifuged (5 min, 1000 rpm) and plated on a 10 cm dish. Four to five such electroporations were performed per construct to ensure the recovery of sufficient number of positive clones. After 24 h, 200 μg/ml G418 (Invitrogen) was added to the medium and the selection continued for 2–3 days, after which 10 μM 6-TG (6-thioguanine or 2-amino-6-mercaptopurine, Sigma-Aldrich) with 200 μg/ml G418 selection was carried out for an additional 6–8 days. Resistant clones were picked in a SNL-seeded 96-well plates and frozen down after reaching confluence. During the picking of clones a replica plate with 0.1% gelatin-coated wells was prepared for Southern Blot screening. For blastocyst injection positive clones were thawed onto a 48-well plate, passaged on 12-well plates, and expanded on 6-well plates before transfer on ice to the EMBL Transgenic Facility.