Journal: Cellular and Molecular Life Sciences

Article Title: DNA methylation regulates the expression of the negative transcriptional regulators ID2 and ID4 during OPC differentiation

doi: 10.1007/s00018-021-03927-2

Figure Lengend Snippet: Targeted methylation of Id2 / Id4 with CRISPR-dCas9 results in lower gene expression and boosts OPC differentiation. A Primary OPCs were transfected with a CRISPR-pdCas9-DNMT3a vector targeting the Id2 or Id4 promoter region. Numbers reflect the number of CpG sites measured for methylation. B Methylation analysis confirms targeted methylation of the promotor of both genes 6 days after transfection, compared to control-transfected cells ( n = 5, two-way repeated measures ANOVA with Šídák's multiple comparisons test). C Gene expression analysis showed lower expression of both genes. Data are corrected for the most stable housekeeping genes ( Rpl13a and Ywhaz ), n = 5, one-sample t test. D Representative images (20 × MBP and DAPI, 40 × MBP) and quantification (fluorescence area and average process length) of OPCs, 9 days post transfection ( n = 5, one-sample t test). E Myelin gene ( Mag, Mbp , Mobp ) expression was increased after targeted methylation of either Id2 or Id4 ( n = 7–8, Wilcoxon test). Data are represented as mean ± SEM, * p < 0.05, ** p < 0.01. sgRNA guide RNA, inactive catalytic inactive pdCas9-DNMT3A-PuroR vector (control), active catalytic active pdCas9-DNMT3A-PuroR vector

Article Snippet: Plasmids were transfected into primary OPCs 24 h after seeding, using the OZ Biosciences NeuroMag Transfection Reagent (Bio-connect, Huissen, The Netherlands), following the manufacturer’s instructions.

Techniques: Methylation, CRISPR, Expressing, Transfection, Plasmid Preparation, Fluorescence