Capn2+/- ES cells were subjected to clonal selection in the presence of 2 mg/mL G418 in attempts to generate homozygous mutant cells by gene conversion. This procedure has been extensively applied to targeted ES cells and was readily achieved in the case of Capn4+/- ES cells [31]. In this case, however, no Capn2-/- ES cells were isolated in screens of 126 drug-resistant clones. The inability to isolate Capn2-/- ES cells, in concert with the absence of Capn2-/- embryos beyond the 8-cell stage, suggested that m-calpain activity might be essential for cell viability or the establishment of viable ES cell clones.