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

Dennis Ek Tan; Baojiang Wu; Siqin Bao; Wee-Wei Tee

doi:10.1016/j.xpro.2022.101684

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

STAR Protoc. 2022 Dec 16;3(4):101684. doi: 10.1016/j.xpro.2022.101684. Epub 2022 Oct 7.

Authors

Dennis Ek Tan¹, Baojiang Wu², Siqin Bao², Wee-Wei Tee³

Affiliations

¹ Chromatin Dynamics and Disease Epigenetics Group, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A∗STAR), Singapore, Singapore.
² Research Centre for Animal Genetic Resources of Mongolia Plateau, College of Life Sciences, Inner Mongolia University, Hohhot, China.
³ Chromatin Dynamics and Disease Epigenetics Group, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A∗STAR), Singapore, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. Electronic address: [email protected].

Abstract

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).

Keywords: Cell differentiation; Developmental biology; Flow cytometry/Mass cytometry; Microscopy; Stem cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Blastocyst
Cell Differentiation
Embryo, Mammalian*
Mice
Mouse Embryonic Stem Cells*