Genetically Engineered Mouse ES Cells

KC-RIK ES cells were cultured in M15 +LIF media and targeted as described previously (Dow et al., 2012 (link); Saborowski et al., 2014 (link)). Clones were selected with hygromycin (Roche) and subjected to functional testing and copy number validation. For functional testing, clones were treated cultured ±Cre expressing Adenovirus (University of Iowa) cultured for 3 days ± doxycycline. Clones that were GFP +as assessed by flow cytometry in the Cre +Dox condition were assessed for single integration into the CHC locus using the Taqman copy number assay for GFP (Life Technologies) on a ViiA7 RT–PCR machine (Life Technologies). Positive clones were expanded and switched to KOSR + 2I 2 days prior to blastocyst injection. The identity and genotype of the ES was authenticated by allele-specific PCRs, and normal genomic status was confirmed by high-resolution Comparative Genomic Hybridization (CGH) array, as previously described (Saborowski et al., 2014 (link)). ES were confirmed to be negative for mycoplasma and other microorganisms before injection.