Effect of depletion of Hp1a (Cbx5) and Hp1ß (Cbx1) on gene expression of mouse embryonic stem cells (mESCs)

To investigate the function of HP1a and HP1ß proteins in the regulation of heterochromatin in mESCs, we isolated blastocysts from matings of mice homozygous for Hp1ßF/F; Hp1aF/F in a single well of a 96-well plate in ESC medium. After 2 days, when blastocysts hatched, they were trypsinized and cells were seeded in the same well for other 2 days. Expanded mESC clones were further tested for their genotype by PCR. A single clone of Hp1ßF/F; Hp1aF/F was transfected with either pCAG-Cre-GFP or pCAG-GFP as a control. GFP positive cells were sorted by flow cytometry and seeded into 10 cm plates. Individual clones were isolated and expanded. Deletion of Hp1ß or Hp1a floxed alleles in pCAG-Cre-GFP transfected clones was tested by Western Blotting with appropriate antibodies. Overall design: We then performed gene expression profiling analysis using data obtained from RNA-seq from 2 different clones with deleted Hp1a and Hp1ß and 1 control clone.