We previously reported that mouse iPSC lines with high chimera-forming ability (Aoi et al., 2008) have DNA methylation profiles close to those of ESCs (Sato et al., 2010).
Because of the small number of EPI cells, a high-ratio chimera could be generated after injection of the same number of pluripotent stem cells into recipient embryos, which increased the ratio of exogenous cells in the ICM.
In fact it was demonstrated that although fewer pups develop to term after morula injection, a higher percentage of them represent high contribution chimeras with higher frequencies of germline transmission compared to injections into blastocyst host embryos [32], [34], [35], [44].
These modified GEMM-ESCs can be used to generate high quality chimeras that are likely equally susceptible to tumor induction as the original GEMM and serve as a defined experimental cohort differing only by the introduced modification.
High percentage chimeras were crossed to C57BL/6 females to establish germline transmission and then crossed to PGK1-FLPo mice [B6(C3 -Tg(Pgk1-FLPo)10Sykr/J, Pgk1-FLPo 10Sykr/J] to remove the Frt-flanked selection cassette (confirmed by PCR).
Furthermore, re-introduction of these cells into the mouse blastocyst led to the formation of high percentage chimeras, indicating their ability to participate in normal murine development, a property not frequently shared with EC cells.
Injection of ESCs into morulae instead of blastocysts (Plagge et al, 2000) leads to very high quality chimeras but also causes an increase in birth problems, still-born animals and pups with growth retardation, in particular for FVB/n and FVB/n;129/Ola ESC clones (Fig 1D and supplementary Table S1).
Clone to clone variation exists both in the ability to derive high-contribution chimeras and the number and quality of chimeras derived from the same cell line in the different media tested (Table S1 and Table S2).
