A PiggyBac-Based Recessive Screening Method to Identify Pluripotency Regulators (original) (raw)
Figure 5
Tcf3 deficiency suppresses serum-induced differentiation.
A. Parental NN97-5 cells differentiate after 4 days in serum without LIF while Tcf3 gene trap mutant P1-2 cells remain undifferentiated and retain uniform Oct4 expression in serum. B. Flow cytometry analysis for Rex1-EGFP positive cells during monolayer differentiation in serum. P1-2, Tcf3 gene trap mutant; CreA12, heterozygous Tcf3 Cre-revertant; CreD10, homozygous Tcf3 Cre-revertant. Graph shown is a representative of two independent experiments. C. Tcf3 mutant (P1-2) and the Tcf3 reverted cells were plated at single cell density in serum with or without LIF for colony forming assay. Colonies were stained after 9 days for alkaline phosphatase (AP) activity and colony numbers were quantified manually. Undifferentiated colonies are showing in red in figure and partially differentiated showing in green and differentiated showing in yellow. D. Images show typical AP positive morphologically undifferentiated ES cell colonies generated by P1-2 cells in serum with or without LIF. The experiment has been repeated once.