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Chimaeric mouse generation and analyses
For production of diploid and tetraploid chimaeras with STAP cells, diploid embryos were obtained from ICR strain females. Tetraploid embryos were produced by electrofusion of 2-cell embryos. Because trypsin treatment of donor samples turned out to cause low chimaerism, STAP spherical colonies were cut into small pieces using a microknife under the microscope, and small clusters of STAP cells were then injected into day-4.5 blastocysts by a large pipette. The next day, the chimaeric blastocysts were transferred into day-2.5 pseudopregnant females. For experiments using STAP cells from CD45+ cells without the Oct4-gfp reporter, STAP cell clusters were identified by their characteristic cluster morphology (they are made of very small cells with no strong compaction in the aggregate). When the STAP conversion conditions (low pH) were applied to CD45+ lymphocytes, most day-7 clusters that were large and contained more than a few dozen small cells were positive for Oct4 (although the expression level varied).
