Fig. 1. Schematic diagram showing the production of NF-pEmbryo. NF-pEmbryos were produced by serial nuclear transfer. ZP-free technology was applied in first nuclear transfer. The haploid chromosome complex (white) obtained by the first nuclear transfer was transferred to MII oocyte in the second nuclear transfer. The reconstituted complex was fused and activated to form diploid NF-pEmbryo with two pronuclei (white: GFP-transgenic mice; black: KM white mice).

Fig. 2. Construction of a diploid NF-pEmbryo. (A) GV stage oocytes from KM white mice (×200). (B) ZP-free GV stage oocytes after pronase digestion (×200). (C) Oocyte with GV enucleated (×200). (D) Ovaries from 1-day old C57BL/6J mice (×100). (E) Non-growing oocytes from 1-day old C57BL/6J mice ovaries under UV light (×200). (F) Enucleated GV oocytes adhered to non-growing oocytes with PHA-P (×200). (G) Oocyte extruding first PB after fusion and maturation (×400). (H) Reconstituted NF-pEmbryos with two pronuclei and two PBs (×200). (I) The NF-pEmbryos under UV light (×200).

Fig. 3. Morphology and karyotype of NF-pES cell colonies. (A) NF-pES cell colonies after 2 days culture (×100). (B) NF-pES cell colonies under UV light (×100). (C) Representative karyotype of NF-pES cell (×1000).

Fig. 4. Character of NF-pES cells derived from reconstituted NF-pEmbryo. (A) The NF-pES cell colony stained positive for alkaline phosphatase (×400). (B) The NF-pES cells stained positive for SSEA-1 (×400). (C) The NF-pES cells stained positive for Oct4 (×400).

Fig. 5. Immunohistological staining of NF-pES cells-derived teratomas. (A) Nestin-positive tissue with neural tube-like structure. (B, F, J, N, R) Expression of GFP under UV light. (C, G, K, O, S) Nuclei stained by Hoechst 33342. (D) Merged image of A, B and C. (E) Desmin-positive tissue with striated muscle-like structure (slit section). (H) Merged image of E, F and G. (I) Desmin-positive tissue with striated muscle-like structure (transverse section). (L) Merged image of I, J and K. (M) Cytokeratin seven-positive tissue with digestion tube-like structure (transverse section). (P) Merged image of M, N and O. (Q) Cytokeratin seven-positive tissue with hepatic cord-like structure (slit section). (T) Merged image of Q, R and S. Magnification: ×400.

Fig. 6. Morphology and immunohistological analysis of EB, derived spontaneously from differentiating pES cells. (A) Phase contrast imaging of EB from NF-pES cells (×40). (B) The same EB under UV light (×40). (C) EB section was positive with ectoderm marker: nestin (red) (×40). (D) EB section was positive with mesoderm marker: desmin (red) (×40). (E). EB section was positive with ectoderm marker: α-fetaprotein (red) (40). Counterstain was with Hoechst (blue).

Fig. 7. Coat color of hybrid and chimaeric mice. (A) Coat color of F1 hybrid mice from KM white and C57BL/6J mice. (B) Coat color of chimeric offspring obtained by NF-pES cells injected into blastocyst cavity of KM white mice.

MII oocytes derived from non-growing oocytes by the GV transfer

Diploid parthenogenetic blastocysts contained genomes from non-growing and fully grown oocytes