Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains

Mouse embryonic stem cells (mESCs) are key tools for genetic engineering, development of stem cell–based therapies and basic research on pluripotency and early lineage commitment. However, successful derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbred mouse strains. Recently, there have been considerable advances in the field of embryonic stem cell biology, particularly in the area of pluripotency maintenance in the epiblast from which the mESCs are derived. Here we describe a protocol for efficient derivation of germline-competent mESCs from any mouse strain, including strains previously deemed nonpermissive. We provide a protocol that is generally applicable to most inbred strains, as well as a variant for nonpermissive strains. By using this protocol, mESCs can be derived in 3 weeks and fully characterized after an additional 12 weeks, at efficiencies as high as 90% and in any strain background.