pyrosequencing-based luminometric dna methylation assay (luma)  (Pyrosequencing Inc)

Journal: Genome Biology

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

doi: 10.1186/s13059-021-02321-2

Figure Lengend Snippet: 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)

Article Snippet: 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).

Techniques: Over Expression, Activation Assay, Stable Transfection, Expressing, Transduction, Control, Western Blot, Phospho-proteomics, Standard Deviation, CpG Methylation Assay, DNA Methylation Assay, Two Tailed Test

Journal: Genome Biology

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

doi: 10.1186/s13059-021-02321-2

Figure Lengend Snippet: 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)

Article Snippet: Since Dusp9 has been suggested to be responsible for the reduction of global CpG methylation levels typically observed in female mESCs (20–30% compared to 60–80% in male mESCs) [ , , ], we analyzed how over-expression of Dusp9 and Klhl13 affected global DNA methylation through the pyrosequencing-based luminometric DNA methylation assay (LUMA; Fig. g).

Techniques: Over Expression, Activation Assay, Stable Transfection, Expressing, Transduction, Control, Western Blot, Phospho-proteomics, Standard Deviation, CpG Methylation Assay, DNA Methylation Assay, Two Tailed Test