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

Abstract

Mouse embryonic stem cells (mESCs) are critical 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 significant advances in the field of embryonic stem cell biology, particularly in the area of pluripotency maintenance in the epiblast from which mESCs are derived. Here we describe a protocol for efficient derivation of germline competent mESCs from any mouse strain, including strains previously deemed non-permissive. We provide a primary method that is generally applicable to most inbred strains, as well as an alternative method for non-permissive strains. 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.

GATA6 Levels Modulate Primitive Endoderm Cell Fate Choice and Timing in the Mouse Blastocyst

“…It has been shown in ES cells that activation of FGF/Erk signaling directly represses Nanog transcription (Santostefano et al, 2012). To test if this down-regulation of NANOG leads to an up-regulation of Gata6 or if FGF signaling can directly influence Gata6 expression, we derived Gata6 +/+ and Gata6 −/− ES cells (Czechanski et al, 2014) and analyzed gene expression after culture in the presence of FGF. While undifferentiated Gata6 +/+ and Gata6 −/− ES cells cultured in LIF exhibited little or no expression of Gata6 , Gata6 transcripts were detected in Gata6 +/+ and Gata6 −/− ES cells maintained in the presence of FGF (Figure 5E).…”

An Eye Organoid Approach Identifies Six3 Suppression of R-spondin 2 as a Critical Step in Mouse Neuroretina Differentiation

“…The generation of this CKO ESCs will not only provide us with an important validation of the organoid culture system as a powerful resource to identify and evaluate candidate genes, but if successful, it will also become a useful approach for other studies in which the use of conditional null ESCs will be the preferred strategy. Although several previous reports described methods for ESC derivation from blastocysts (Czechanski et al, 2014; Gong et al, 2006; Kawase et al, 1994; Lee et al, 2012; Suzuki et al, 1999), their efficiency was not high enough to consider them as an alternative for the derivation of CKO ESCs. Furthermore, none of those protocols derived ESCs from mixed genetic backgrounds (in our case mixed C57BL/6J/NMRI).…”

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation

“…Neuro-2a (N2a) cells were originally from Sigma-Aldrich and cultured with the same medium. Cas9 mouse embryonic stem cell (Cas9 mESCs) lines were derived from blastocysts of homozygous Rosa26-Cas9 knock-in mice using previously described procedures (Czechanski et al, 2014). Cells were then maintained in N2B27 2ILIF media on Matrigel (Cultrex) coated plates.…”