Fig. 1.
Mouse EG cells can be maintained and derived in 2i-LIF. (A) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells. (B) Phase contrast and fluorescence images of Oct4-ΔPE-GFP EG cells cultured in: FCS-LIF on a MEF feeder layer; FCS-LIF after one passage without feeders; 2i-LIF without feeders. (C) Flow cytometry analysis of Oct4-ΔPE-GFP EG cells cultured in 2i-LIF compared with FCS-LIF on feeders. Gates were set to eliminate feeders and cell debris. (D) Colony-forming assay on Oct4-ΔPE-GFP EG cells. Colonies were scored for alkaline phosphatase (AP) and designated positive, negative or mixed. Data are means of two biological replicates. (E) Schematic of EG cell derivation protocol. On day 3, medium was changed to either FCS-LIF or 2i-LIF. (F) Comparison of GFP-positive EG cell colony formation following addition of 2i-LIF on day 3 compared with FCS-LIF. (G) High-contribution coat colour chimaeras generated with agouti EG cells derived in 2i-LIF injected into C57BL/6 blastocysts. (H) Chimaera, C57BL/6 mate, and mixed litter of agouti and black pups. Scale bars: 100 μm in A; 25 μm in B.