181 MOUSE TRIPLETS DEVELOPED FROM SINGLE BLASTOMERES OF AN 8-CELL-STAGE EMBRYO SUPPORTED WITH TETRAPLOID EMBRYOS

Abstract

Chimera production using altered ES cells became a key tool for generating transgenic mice. However, chimeras are more than just a tool for making mouse mutants; they play a crucial role in analyzing the biological effects of genetic changes. Chimeras can be made by combining two whole 8-cell embryos or by combining subsets of blastomeres of two cleavage stage embryos. Because, at these stages, the early embryonic cells are not yet restricted in their lineage potency, they are equally capable of contributing to the inner cell mass or the trophectoderm. Pluripotency of single blastomeres of the 4-cell and 8-cell mouse embryo has been proved indirectly by aggregating them with carrier blastomers of a different genotype, giving rise to chimeric blastocyst (Tarkowski et al. 1967 J. Embryol. Exp. Morphol. 18, 155-180). In our study we wanted to demonstrate that a single blastomere of an 8-cell stage embryo, supported with tetraploid embryos at the 4-cell stage, is capable of developing into a healthy animal. When tetraploid embryos are used to make chimeras together with diploid cells, tetraploid cells rarely contribute to the embryo itself, but contribute mainly to the primitive endoderm and the trophectoderm, so in this case the newborns will be derived only from the diploid blastomers used (Nagy et al. 1990 Development 110, 815-821). We produced chimeras by aggregating a single blastomere [(2n)(1-cell)] derived by combining sexed, GFP-expressing, diploid 8-cell stage embryo (Hadjantonakis et al. 1998 Nat. Genet. 19, 220-222) with either a sexed diploid 7-cell embryo [(2n)(7-cells)] (one of the 8-cells was taken for sexing) or a non-sexed tetraploid embryo [(4n)(4-cells)]. The aggregates were cultured in vitro and transferred as blastocysts to the uteruses of pseudo-pregnant females. Fetuses were removed by Caesarian section and raised by lactating foster mothers. From the transferred 84 [XY(2n)(1-cell)]/[XX(2n)(7-cells)] aggregates we obtained 12 (14.3%) newborns, 11 (91.7%) males and one (8.3%) female. From the transferred 27 [XY(2n)(1-cell)]/[(4n)(4-cells)] aggregates, where one XY blastomere was combined with a tetraploid embryo, we obtained 7 (25.9%) male newborns: 1 triplet and 2 pairs of twins. We also transferred four [XX(2n)(1-cell)]/[(4n)(4-cells)] chimera embryos, where an XX-blastomere was aggregated with a tetraploid embryo; we obtained a set of living female triplets (75%) from this aggregate. We demonstrated that a single blastomere of the 8-cell-stage embryo is capable of developing into a living newborn. We obtained identical pups from three blastomeres isolated from the same embryo (triplets). This way we produced single blastomere clones and could control the sex of the new generation. This research was supported by grants from OTKA T037582 and RO-1/2002 Intergovernmental S&T cooperation program.