Table 1.
Primer Sequences.
Table 2.
Primer sequence in COBRA.
Fig 1.
Production and Characterization of Tetraploid Embryonic Stem Cells (TESCs).
(A) Diagram of production of TESCs with elecrofused diploid embryos. Two or three embryos were seeded onto a mouse embryonic fibroblast (MEF) feeder layer. (B) Flow cytometry analysis of DNA content after 10 passages. A representative figure is shown. (C) Karyotyping analysis of metaphase chromosome spreads. Tetraploid ESCs normally had 80 chromosomes, while control diploid ESCs had 40 chromosomes at passage 10. A representative figure is shown. (D) Proliferating TESCs formed typical round-shaped mouse ESC colonies with clear boundaries similar to control ESCs. TESC colonies also stained positive for the control ESC-positive marker alkaline phosphatase (ALP). A representative figure is shown. (E) Growth rates. The relative cell number was based on the cell number at day 0 (2 × 104 cells). All data represent the mean and SEM (n = 3). *P < 0.001. Scale bar, 100 μm.
Fig 2.
Expression and Epigenetic Analysis of TESCs.
(A) Relative expression of pluripotency markers by quantitative real-time RT-PCR analysis at passage 10. All data represent the mean and SEM (n = 3). *P < 0.05. (B) Analysis of DNA methylation profiles. DNA methylation levels in Oct3/4 and Nanog genes were determined by Bio-COBRA in TESCs and ESCs. All data represent the mean and SEM (n = 3). *P < 0.05. (C) Immunostaining of pluripotency markers. DAPI was used to stain DNA. A representative figure is shown. Scale bar, 50 μm.
Fig 3.
The Differentiation Potential of TESCs.
(A) Morphology of embryoid bodies generated from TESCs and ESCs. Scale bars, 100 μm. (B) Relative expression of various cell lineage marker genes in embryoid bodies generated from TESCs and ESCs by quantitative real-time RT-PCR analysis. All data represent the mean and SEM (n = 3). *P < 0.05. (C) Teratoma formation by TESCs. Scale bars, 50 μm.
Fig 4.
Development of Morula Embryo Aggregates from Injections of In Vitro Cultures of EGFP-Expressing TESCs.
Morphology, fluorescence, and merged images of aggregated embryos at the blastocyst stage.