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Fig. 3 | Genome Biology

Fig. 3

From: Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

Fig. 3

Over-expression of Klhl13 and Dusp9 in male mESCs leads to an enhanced pluripotency state and slower differentiation kinetics. a Schematic representation of the dCas9-SunTag system used for gene activation. b–e To over-express Dusp9 (yellow) and Klhl13 (blue), male E14 mESCs, stably expressing the doxycycline-inducible SunTag system, were either transduced with one of two different sgRNAs targeting the respective promoter regions or with non-targeting control (NT) sgRNAs and were treated for 3 days with 1 μg/ml doxycycline as indicated. Protein levels of Dusp9 (left) and Klhl13 (right) were quantified via immunoblotting (b), expression levels of MAPK target genes Spry4 and Egr1 (c) and of naive pluripotency factors Nanog and Prdm14 (e) were assessed by qPCR and phosphorylation of Mek and Erk was quantified by immunoblotting (d). The immunoblot signals were normalized to Tubulin (b) or to total Mek/Erk (d) and to the mean of two doxycycline-treated non-targeting control sgRNAs. qPCR measurements were normalized to two housekeeping genes and to the respective untreated control (−Dox). Dots and triangles depict individual measurements of the two different sgRNAs, and thick bars show the mean of three biological replicates. f Dusp9- and Klhl13 over-expressing mESCs were treated with 1 μg/ml doxycycline 24 h before differentiation via LIF withdrawal for 4 days, and expression levels of pluripotency factors were measured by qPCR at different time points as indicated. Mean and standard deviation across 3 biological replicates is shown. g Global CpG methylation levels in cell lines over-expressing Dusp9 and Klhl13 via doxycycline treatment for 3 passages were assessed via pyrosequencing-based luminometric DNA methylation assay (LUMA). *p < 0.05 in a two-tailed paired Student’s t test comparing the Dusp9/Klhl13 over-expressing samples and the non-targeting controls (mean of sgRNA1 and sgRNA2)

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