Derivation of mouse embryonic stem cells

Abstract

Here we describe a simple and efficient protocol for derivation of germline chimera-competent mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) blastocysts. The protocol involves the use of early-passage mouse embryonic fibroblast feeders (MEF) and the alternation of fetal bovine serum- and serum replacement (SR)-containing media. As compared to other available protocols for mESCs derivation, our protocol differs in the combination of commercial availability of all reagents, technical simplicity and high efficiency. mESC lines are derived with approximately 50% efficiency (50 independent mESC lines derived from 96 blastocysts). We believe that this protocol could be a good starting point for (i) setting up the derivation of mESC lines in a laboratory and (ii) incorporating further steps to improve efficiency or adapt the protocol to other applications. The whole process (from blastocyst extraction to the freezing of mESC line) usually takes between 15 and 20 d.

Chromosome number variation in three mouse embryonic stem cell lines during culture

“…Although mESC lines have been established many years ago (Evans and Kaufman 1981;Martin 1981) new culture conditions need to be developed to maintain the correct chromosome complement in long-term culture, for example using serum-replacement media (Wiles and Johansson 1999;Fletcher et al 2006;Bryja et al 2006;Amit and ItskovitzEldor 2006) and alternative methods for cell dissociation as applied for hESC lines (Suemori et al 2006). The results of our study underline the importance of a cytogenetic analysis to further our understanding of the biology of mESCs and for the improvement of the culture conditions needed to maintain a stable chromosome complement.…”

rRNA Genes Are Not Fully Activated in Mouse Somatic Cell Nuclear Transfer Embryos

“…Cell Culture and Treatment-Derivation and culture of mouse ESCs were according to a previous protocol (22). Cells were cultured in DMEM containing 15% FBS, 50 g/ml penicillin/streptomycin (Invitrogen, 15140-148), 100 M nonessential amino acids (Invitrogen, 11140-050), 100 M ␤-mercaptoethanol (Sigma, M7522), and 1000 units/ml leukemia inhibitory factor (Chemicon, ESG1107).…”

Derivation of androgenetic embryonic stem cells from m-carboxycinnamic acid bishydroxamide (CBHA) treated androgenetic embryos

“…Androgenetic embryonic stem cell lines were derived as previously described [19]. Blastocysts were transferred to gelatinized 4 multi-well plate coated with mitomycin C-treated murine embryonic fibroblast feeder (MEF) cell layers in ES medium.…”