Oct4+/− Cells Express an Elevated Level of NANOG and Lack a Nanog-Negative Population
(A) Immunoblot analysis of Oct4+/+ (E14Tg2a, CGR8, and ZIN40) and of Oct4+/− (OKO160 [] and ZHTc6 [cultured in 1 μg/ml doxycycline []) cell lines. See for a detailed summary of all cell lines used in this study. ZHBTc4 (Oct4−/−) cells (), which express an Oct4 transgene at heterozygous levels (when cultured without doxycycline) also showed increased Nanog. The parietal endoderm cell line D7A3/PE () provides a negative control.
(B) Quantitative PCR (qPCR) analysis of Oct4 and Nanog mRNAs in Oct4+/+ (E14Tg2a and CGR8) and Oct4+/− (OKO160 and ZHTc6 [cultured in 1 μg/ml doxycycline]) cells. Error bars represent SD; n = 3.
(C) Immunofluorescence analysis of wild-type (WT; E14Tg2a) and Oct4 mutant (OKO160, ZHTc6 [cultured in 1 μg/ml doxycycline], and ZHBTc4 [cultured without doxycycline]) cells for Nanog (green) and Oct4 (red).
(D) Quantitative analysis of Oct4 protein levels in individual cells in colonies of Oct4+/+ (E14Tg2a, green) and Oct4+/− (OKO160, brown) cells.
(E) Intracellular FACS quantitation of Oct4 and Nanog protein levels in individual cells of Oct4+/+ (E14Tg2a, red) and Oct4+/− (OKO160, blue) cultures.
(F) Top, immunofluorescence analysis of blastocyst expression of Oct4 protein following the aggregation of GFP-marked Oct4+/− ESCs with WT morula. Bottom, quantitation of immunofluorescence for Oct4 in the ICM for WT and GFP-marked Oct4+/− cells.
(G) FACS analysis for Nanog:GFP in Oct4 WT (Tg2a-Nanog:GFP) and Oct4 mutant cells (OKO-Nanog:GFP and ZHTc-Nanog:GFP [cultured in 1 μg/ml doxycycline] derived from the parental lines OKO160 and ZHTc6, respectively; Oct4 genotypes are indicated) at the indicated times following release from puromycin selection (day 0). The percentage of cells in Nanog-low, Nanog-middle, and Nanog-high populations are shown (red) with the coefficient of variation (CV) for Nanog:GFP indicated.
See also and and .