Totipotency Validaiton Of The Mouse Induced Pluirpotent Stem Cells By8-cells Stage Embryo Injection

Production of chimeric and tetraploid embryo complementation mice is the strictest standard tovalidate whether totipotency of a given cell line. The trophoblasts used in this study were CF1mouseembryo fibrolast cells and recipent embryos were derived CD1mouse embryos, while iPS cells originedfrom C57mouse which carries a green fluorescent protein (EGFP) expressing through Oct4promoter, wereused as donor cells. When recipent embryos developed to8-cell stage, we injected a single donor iPS cellexpressing green fluorescent into CD1embryos to achieve chimeric embryos. Blastosphere chimeric ratewas examined by comparison of the fluorescence area and total area of inner cell mass (ICM) whenchimeric embryos were cultured to blastosphere in vitro. Through the embryo transfer (ET), we furtherplanted the chimeric blastocysts into the uterus of the pseudopregnancy mice. By AP staining, fluorescenceappraisal, in vitro embryoid body identification and karyotype analysis, we showed that this iPS cell linepassaged from28to34generation still had definite totipotency. To prepare chimeric embryos,497embryoswere injected with the iPS cell. For2n embryos (274),183of them developed to blastosphere, showing arate of66.8%; For4n embryons (223),116pieces developed to blastosphere with a rate of52.0%. Based onthe area ratio, we randomly chose10embryos from each of chimeric embryos (2n/2n and2n/4n) tocalculate blastosphere chimeric rate, which was above70%(2n/2n embryos:86.0%±8.4%, P=0.05;2n/4nembryos:70.4%±18.1%, P=0.05) on average. The above blastocysts have been already planted into thepseudopregnancy mice, but no chimeric mice were born yet. In summary, our data showed that:1,8-cellstage single-celled injection method could greatly improve the blastosphere chimeric rate.2, embryoidbodies formed by mouse iPS cells (IP2-19) contained a uniform distribution of totipotent cells which still maintained the Oct4gene expression.