Fig. 2. The aggregation procedure using Sp^2H and Sp^2G embryos. The scheme for one of the aggregation procedures using Sp^2H and the allele generated by knocking-in the lacZ gene into the Pax3 locus. Briefly, heterozygous animals (Sp^2H/+ and Sp^2G/+) were intercrossed and morulae were prepared at E2.5 of gestation. In parallel, embryos from NMRI (albino) wild-type intercrosses were also prepared. Embryos from both matings were denuded from the zona pellucida using acidic tyrode solution (Hogan et al., 1994) and subjected to aggregation as indicated. The embryos from Sp^2H and Sp^2G crosses are on a mixed 129/sV and C57Bl/6 background and presented in gray. The embryos were left to develop overnight to blastocysts and subsequently transferred to foster mothers. Chimeric embryos were prepared at the appropriate time. DNA was prepared from embryonic membranes and both alleles are detected by PCR. Embryos that are positive for both alleles are supposed to contain homozygous Pax3^-/- cells. The embryos that are positive only for lacZ are considered to consist of wild-type and Pax3^+/- cells and are used as controls. In older embryos, the degree of chimerism is also monitored by eye pigmentation.