Generation of 2C-like mouse embryonic stem cells in vivo to evaluate developmental potency

This protocol describes an optimized workflow for generating 2-cell (2C) stage-like mouse embryonic stem cells (mESCs) for microinjection into 8-cell stage mouse embryos to evaluate the developmental potency of these cells. We detail the following steps: 1) chemical suppression of glycolysis to induce a 2C-like state in mESCs, 2) flow cytometry to enrich for 2C-like cells, 3) embryo microinjection of 2C-like mESCs into 8-cell stage mouse embryos, and finally, 4) immunofluorescence staining of the chimeric blastocysts.For complete details on the use and execution of this protocol, please refer to Hu et al. (2020).