Journal: Development (Cambridge, England)
Article Title: Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state
Figure Lengend Snippet: 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.
Article Snippet: FCS-LIF conditions comprise DMEM-F12 medium supplemented with 15% FCS, 0.1% MEM non-essential amino acids, 4 mM glutamate, 2 mM sodium pyruvate, 0.1 mM 2-mecaptanethanol and recombinant mouse LIF (1200 U/ml; ESGRO, Chemicon) or human recombinant LIF generated in-house by transient transfection of Cos7 cells with plasmid encoding human LIF.
Techniques: Derivative Assay, Fluorescence, Cell Culture, Flow Cytometry, Cytometry, Generated, Injection