Extended Data Figure 3: Characterization of knockout ES cell mutants and quantification of human iPS cell reprogramming efficiency.
From: Co-repressor CBFA2T2 regulates pluripotency and germline development
a, b, Strategy for generating Prdm14- and Cbfa2t2-knockout (KO) mES cells via CRISPR–Cas9 genome editing. Sequencing chromatograms confirming homozygous disruption of the locus are depicted. c, Cbfa2t2- and Prdm14-knockout ES cells require 2i to maintain growth. ES cell lines generated under FBS plus LIF plus 2i conditions were continuously cultured in FBS plus LIF plus 2i (top, middle), or switched to FBS plus LIF (bottom). Eight days after 2i withdrawal (FBS plus LIF), well-formed ES cell colonies were undetectable; instead, mutant ES cells appeared to be differentiated. Scale bar, 100 μm. d, Proliferation rates of wild-type (WT) and mutant knockout ES cells as described in c. Data were obtained from three biological replicates. Please note error bars shown in the plots. Owing to the logarithmic scale used here, some error bars are very small and might be invisible. e, RNA-seq MA plot (log ratio (M) versus mean average (A)) in the indicated ES cells. Data are representative of three biological replicate experiments for each line. Mean abundance is plotted on the x axis and enrichment (both in log2 scale) is plotted on the y axis. Genes depicted in red are differentially expressed with a FDR < 0.0001. f, Heat map showing relative expression of all differentially expressed genes as described in Fig. 2c. The only difference is now the heat map is centred on CBFA2T2 differentially expressed genes, rather than PRDM14 differentially expressed genes. g, Scheme of human fibroblast reprogramming to iPS cells. Fibroblasts were transduced with lentiviruses expressing polycistronic OCT4/KLF4/SOX2/c-MYC (OKSM) and either PRDM14 or CBCFA2T2. Three weeks later, bright-field images of successfully reprogrammed colonies (left) and live TRA-1-81 staining (right) were recorded. Scale bar, 500 μm. h, Quantification of human iPS cell reprogramming efficiency based on TRA-1-81 staining with secondary antibody conjugated with horseradish peroxidase (HRP) and substrate DAB. Error bars are based on four biological replicates of each condition. The source data are included in the Supplementary Information.