Haploid_ES_cell_sequencing

HAP-3 mouse ES cells, which are derived from a 129sv genetic background, were mutagenised in their haploid state using ethylmethanesulfonate (EMS) at doses of 200, 400, 500, 600, and 800 ug/ml. After mutagenesis cells were grown for 4 days in the presence of 2 uM 6-thioguanine (6-TG) to select for clones carrying mutations in HPRT. As part of this project we have sequenced the un-mutagenised HAP-3 cell line, and seven 6-TG resistant colonies selected from clones generated at different EMS doses. Our aim is to determine the number of mutations generated in the genome of HAP-3 cells when EMS is used as a mutagen. Since mutations in mismatch repair genes (such as MLH1 or MSH2) may give rise to 6-TG resistance, mainly by increasing the frequency of mutations at the HPRT locus, we will also look for mutations in these genes.