Cell Lines

All mouse embryonic stem cell lines were expanded under the 2i+LIF conditions (Ying et al., 2008), in a humidified incubator at 37°C and 7% CO₂, and routinely tested negative for mycoplasma infection. A male, karyotypically normal, tdTomato-expressing mouse embryonic stem cell line was derived from E3.5 blastocysts obtained by crossing a male ROSA26tdTomato (Jax Labs – 007905) with a wildtype C57BL/6 female. Competence for chimera generation was assessed using morula aggregation assay. Targeting of the T locus was performed using the CRISPR/Cas9 system (see Method Details), mutant clones were assessed by next-generation sequencing (see Figure S4). Two mutant clones were used to generate T^-/- embryonic chimeras.