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Fig. 2. Observation of Rex1-GFP/Oct3/4-CFP double knock-in ES
cells by microscopy and flow cytometry. (A-H) Morphology and
fluorescence of colonies of
Oct3/4+/Rex1+ and
Oct3/4+/Rex1- cells in culture of
Rex1-GFP/Oct3/4+-CFP double-knock-in ES cell line under
puromycin selection (selection for cells expressing Oct3/4). (A,C,F)
GFP fluorescence, (D,G) CFP fluorescence and (B,E,H) bright field. (A,B) Low
magnification of knock-in ES cells. A small number of GFP
(Rex1)-negative cells was observed. Arrows in B indicate colonies of
Rex1-/Oct3/4+ (GFP-) cells. (C,D,E)
High magnification of colonies in which Rex1+ cells were
dominant. (F,G,H) High magnification of colonies in which
Rex1- cells were dominant. (I,J) Result of analysis of
knock-in cells by flow cytometry. (I) Comparison of GFP fluorescence of
Rex1-knock in cells with the parental cell line (OLC2-1). (J)
GFP fluorescence of Rex1 knock-in cells disappeared within 3 days
when differentiated by withdrawal of LIF. (K) Rex1 knock-in
cells were stained with anti-PECAM-1 monoclonal antibody conjugated with PE
and analyzed by flow cytometry. Most GFP-positive cells reacted with PECAM-1
antibodies. Scale bars: 50 µm.
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