Generation of mouse chimeras from ESCs

ARC/s and DsRed.T3 mice were purchased from the Australian Animal Resources Centre and maintained as homozygous breeding pairs. ESC clones with monoallelic frameshift mutations and the parental A2LoxCre ESC line were used to generate chimeras. Embryo injections were performed as previously described (Sibbritt et al., 2019). Briefly, 8–10 ESCs were injected per eight-cell DsRed.T3 embryo (harvested at 2.5 dpc from super-ovulated ARC/s females crossed to DsRed.T3 stud males) and incubated overnight. Ten to 12 injected blastocysts were transferred to each E2.5 pseudo-pregnant ARC/s female recipient. E9.5 and E11.5 embryos were collected 6 and 8 days after transfer to pseudo-pregnant mice. Embryos showing red fluorescent signal indicating no or low ESC contribution were excluded from the phenotypic analysis. Animal experimentations were performed in compliance with animal ethics and welfare guidelines stipulated by the Children's Medical Research Institute/Children's Hospital at Westmead Animal Ethics Committee, protocol number C230.