Fig. 5. Strategy to generate female mouse chimeras. (A) Schematic diagram of female chimera analysis. Lim1^–/–;Rosa26^tg/+ XO ES cells were generated from Lim1^–/–;Rosa26^tg/+ XY ES cells (Shawlot et al., 1999) by spontaneous loss of the Y chromosome by screening with an Y-chromosome-specific repeat probe, Y353/B. These XO ES cells were injected into XX blastocysts to generate chimeric female mice. XX blastocysts can be distinguished from XY blastocysts by using X-linked ubiquitous GFP male mice for breeding. Chimeric mice were recovered at different stages of embryogenesis and processed for ß-gal staining to visualize Rosa26-marked ES cell-derived XO cells. The yolk sac was collected for reconfirming sexes of injected blastocysts by PCR genotyping for the Sry gene. (B) Southern blot analysis of XO ES cells. Genomic DNA of XO ES cells was blotted after EcoRI digestion and hybridized with a Lim1 probe. The same blotted membrane was hybridized again with Y-chromosome-specific Y353/B probe. Genomic DNA from male and female tails was used for comparison. Note that these XO cells show the same pattern as female tail.