How was the quality control done for the ES cell clones?
We performed multiple-step quality controls for ES cell clone selection. Venus expression was analyzed by both visual fluorescence microscopy and flow cytometry. Relative levels of exogenous transcripts were determined by qPCR using open reading frame primers. Additionally, ES cell clones were genotyped to confirm correct Cre-mediated recombination and to ensure the absence of random integration of the exchange vector and the Cre-expressing vector. ES cells were karyotyped at passage 25, and all clones had normal euploid ranges (64%-96%). All clones passed mycoplasma contamination testing. To minimize clonal variation of gene expression, passage numbers were kept consistent across cell lines. Each transfection was begun at passage 17, and microarray analysis was performed at passage 25. Stock ES cells were cultured on MEFs with the addition of LIF, but in vitro experimental cells were cultured on feeder-free gelatin-coated plates.
