Figure 3.

2i and KOSR ESCs Can Form 100% ESC-Derived Mice When Injected into 2C Embryos

ESCs were derived (D) in and cultured (C) under various conditions; D and C in serum, D in serum and C in 2i, D and C in 2i, D in 2i and C in serum, and D and C in KOSR. Single ESCs from each of these conditions were injected into 2C embryos.

(A) Graph showing the correlation of serum-, 2i-, and KOSR-cultured ESCs with published microarray data of oocytes and 2C embryos (Macfarlan et al., 2012).

(B) Flow cytometry profiles for Zscan4-Emerald ESCs, immunostained for the marker of undifferentiated ESCs PECAM-1, cultured in serum, 2i, and KOSR.

(C) Representative 2C embryos immediately after injection with a single ESC expressing a constitutive H2B-Tomato marker.

(D) Confocal optical sections of an immunostained late blastocyst resulting from the injection of a single 2i-cultured ESC, expressing a constitutive H2B-Tomato marker, into a 2C embryo.

(E) Representative images of litters containing 100% ESC-derived agouti mice generated from the injection of a single 2i- or KOSR-cultured cell into a 2C embryo, along wild-type C57BL/6 littermates. Asterisks indicate mice with ESC contribution.

(F and G) Single ESCs were injected into 2C embryos and their contribution to full-term chimeras assessed according to coat color. (F) Graph showing the percentage of chimeras born out of the total number of pups and quantification of coat color chimerism. n Values are provided above bars and refer to the number of independent ESC clones that were tested for each condition. Error bars indicate SEM. (G) Representative images of full-term mice generated from the injection of single-cell ESCs, D and C in serum (upper panel), or D and C in 2i (lower panel), into 2C embryos. Asterisks indicate mice with ESC contribution.

(H) Graph showing the percentage of chimeras born out of the total number of pups and quantification of coat color chimerism. n Values are provided above bars and refer to the number of independent ESC clones tested for each condition.

(I) ESCs were pre-cultured in serum, 2i, or KOSR for three passages and then switched to XEN cell medium for two passages. Images show representative colonies.

(J) Quantification of the average proportion of colonies under each condition expressing GATA6. A distinction was made between colonies with >20 GATA6⁺ cells and those with <20 GATA6⁺ cells. Error bars indicate SD of the mean of five randomly selected fields of view.

See also Figure S3.