Supplementary Figure 4: Oct4A-null oocytes reprogrammed somatic cell nuclei to pluripotent status.

(a) Immunocytochemistry of E4.0-NT blastocysts show activation of Nanog and Oct4 expression by Oct4A-knockout (Oct4A KO) oocytes. WT, wild type; NT: Nuclear transferred; PA: parthenogenic. (b) Gene expression profiling of NT blastocysts revealed activation of expression of the pluripotent genes Oct4 and Nanog without maternal Oct4A expression. The gene expression levels were obtained with pools of 3 blastocysts with triplicates and presented in comparison with ES cells (ES). Ctr: NT embryos using wild-type oocytes; KO, NT embryos using Oct4A-knockout oocytes; PA: parthenogenic embryos using Oct4-knockout oocytes. The number (1, 2 or 3) right after the abbreviation (Ctr and KO) refers to the biological replicates. (c) Morphology of NT-ES cells grown on MEFs expressing CAG–mRFP and Oct4-GFP.(d) Histology of teratoma from NT-ES cell line RG6 4 weeks after injection into SCID mice as assessed by haematoxylin and eosin staining. The teratoma contained cells of all 3 embryonic germ layers. Upper left panel: keratinized stratified squamous epithelial cells (ectodermal); upper right panel: neural rosettes (ectodermal); lower left panel: striated muscle (mesodermal); lower right: ciliated columnar epithelial cells adjacent to pancreatic acinar cells (both endodermal). (e) A litter of neonatal NT-ES cell-derived pups delivered by cesarean section on E19.0. In this particular litter, 8 pups showed normal full-term development, of which one was dead and one failed to initiate breathing (*). The scale bars represent 30 μm in a, 100 μm inc and 50 μm in d. Values represent mean±S.D. of 3 biological replicates.