Neg513h250uc

PB‐TET vectors containing key germ cell factors Blimp1, Tfap2c and Prdm14 (Nakaki et al, 2013 (link)) were kindly given by F. Nakaki. Cells were transfected with 3 µg each of PB‐TET vectors, pPBCAG‐hph and a PiggyBac transposase vector using the AMAXA Mouse Embryonic Stem Cell Nucleofector kit (LONZA, VPH‐1001). Transfected cells were selected with 200 μg/ml hygromycin B Gold (Ibian Tech., ant‐hg‐1) for 10 days and genotyped by PCR for transgenes. The primer sequences are shown in Table 1.
Copy number integration was estimated by Southern blot hybridization. Briefly, 15 µg of genomic DNA were digested with BamHI. DNA fragments were electrophoresed in 0.8% agarose gel and transferred to an Amersham Hybond XL membrane (GE Healthcare, RPN303S). The b‐geo probe was designed downstream of the BamHI site, obtained by digesting the PB‐TET‐Avi‐Blimp1 plasmid with CpoI/SmaI, labelled with dCTP [α‐32P] (Perkin Elmer, NEG513H250UC) using High Prime (Roche, 11585592001), purified with an Illustra ProbeQuant G‐50 Micro Column (GE Healthcare, 28903408) and hybridization performed in Church buffer. Radioisotope images were captured with a Phosphorimager Typhoon Trio.

Genomic DNA (10 µg) was digested with appropriate restriction enzymes overnight. Subsequently, genomic DNA was separated on a 0.8% agarose gel and transferred to an Amersham Hybond-XL membrane (GE Healthcare, RPN303S). Probes were synthesized by PCR amplification and labelled with dCTP, [α-32P] (Perkin Elmer, NEG513H250UC) using High Prime (Roche, 11585592001) and hybridization performed in Church buffer.