Production of identical mouse twins and a triplet with predicted gender

The aim of this study was to develop a method to generate identical twins and triplets with predicted gender. As a first step toward that aim, single blastomeres obtained from EGFP expressing eight-cell stage embryos and either diploid or tetraploid host embryos were used to compose chimera. We could follow the fate of EGFP expressing diploid blastomere derived cells in 3.5- and 4.5-day-old chimera embryos in vitro. We found that the diploid blastomere-derived cells had significantly higher chance to contribute to the inner cell mass if tetraploid host embryos were applied. After that, we developed a quick and reliable multiplex PCR strategy for sex diagnosis from single blastomeres by simultaneous amplification of the homologous ZFX and ZFY genes. By composed chimeras using single blastomeres, derived from sexed eight-cell stage embryos and a tetraploid host embryo, we could get preplanned sex newborns, wholly derived from these blastomeres. Among these mice, identical twins and a triplet were identified by microsatellite analysis. Unlike clones produced by nuclear transfer, these mice are identical at both the nuclear as well as mitochondrial DNA level. Therefore, the tetraploid embryo complementation method to produce monozygotic twins and triplets could be a valuable tool both in biomedical and agricultural applications.