Increased gp130 signaling in combination with inhibition of the MEK/ERK pathway facilitates embryonic stem cell isolation from normally refractory murine CBA blastocysts.
The standard method for isolation of ES cells from strain 129 mice does not give rise to ES lines of CBA origin. We investigated the effect of inhibition of MEK/ERK signaling in combination with increased stimulation of gp130 signaling on derivation of ES cells from CBA blastocysts. Inhibition of MEKI and MEKII using the drug U0126 and stimulation of gp130 signaling by elevating the level of LIF present gave rise to ES-like lines in 22.6% of explants. No lines arose when MEK was inhibited in the absence of additional LIF stimulation, nor when additional LIF stimulation occurred in the absence of MEK inhibition. Typically for ES cell lines, CBA-derived cells contributed to chimeric mice and differentiated broadly in vitro. Increased levels of gp130 signaling led to similar levels of STAT3 activation in strain 129 and CBA ES cells. We conclude that CBA ES cells have a requirement for additional STAT3 activation.
Roslin Institute, Roslin, Midlothian EH25 9PS, U.K.
Peter
P
Lodge
Jim
J
McWhir
Edward
E
Gallagher
Helen
H
Sang
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001755:Blastocyst / Q000378:metabolism; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D050822:Cytokine Receptor gp130; D004268:DNA-Binding Proteins / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D053605:Leukemia Inhibitory Factor; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008808:Mice, Inbred CBA; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology*; D015534:Trans-Activators / Q000378:metabolism
D015703:Antigens, CD; D002070:Butadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C494200:Il6st protein, mouse; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008562:Membrane Glycoproteins; D009570:Nitriles; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D015534:Trans-Activators; C113580:U 0126; D050822:Cytokine Receptor gp130; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases