Whole_exome_analysis_of_ENU_mutagenized_mouse_ES_cell_lines. Whole_exome_analysis_of_ENU_mutagenized_mouse_ES_cell_lines

Chemical mutagens such as N-ethyl-N-nitrosourea (ENU) are one of the most powerful mutagens together with insertional mutagens (e.g. retrovirus and transposons) and have been extensively used in mouse in vivo mutagenesis programs. ENU has also been used to mutagenize mouse ES cells and proved its efficiency of mutagenesis. An optimal mutagenesis condition can give rise to Hprt mutant in 1 in 1,000. Given that the total number of genes is 30,000, it can be assumed that each cell contains approximately 30 functional mutations. Yet, the exact number of functional mutations and an actual mutation load have not been investigated because of the lack of methods of genome-wide analysis. Recent developments of next-gen sequencing and sequence capture technologies open up a new opportunity to investigate an exact mutation load of ENU mutagenesis. To this end, we are planning to sequence exomes of 9 ENU-mutagenized ES cell clones and one parental ES cell clone. These mutagenized cells were obtained from a Blm-deficiency-mediated phenotype-based screen, in which genes involved in GPI-anchor biosynthesis pathway were targeted [Nature 2004]. All mutants carry mutations in such genes and point mutations were already mapped, which can serve as good positive controls for exome sequencing. Insertional mutagens are also efficient mutagens and identification of insertion sites or mutated genes is extremely easy. However, retroviruses are known to have strong integration hotspots. A bank of mutant ES cells mutagenized by retrovirus possesses over 350,000 clones, 10-times coverage of a whole genome, but mutants only in 10,433 unique genes could be found. Recent our data of transposon integration sites in ES cells also imply hot- and cold-spots of integrations although their coverage is much better than retrovirus. Taken together, whole genome coverage by these insertional mutagens in ES cells may not be possible and an unbiased nature of ENU mutagenesis would be ideal. Difficulties of identification of ENU-induced causal mutations may be no longer problems at the era of sequencing. This proposed project will examine a possibility of ENU mutagenesis in ES cells at an unprecedented depth of analysis. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/