Timed matings and embryo collection

For timed pregnancies, (CBA × C57BL/6)/F1 females (6–26 weeks old) were mated overnight with 23GFP transgenic males (6–52 week old, maintained on a mixed (CBA × C57BL/6) background. Embryos were collected in phosphate-buffered saline (PBS; Gibco, Invitrogen) supplemented with 10% fetal calf serum (FCS; batch tested and obtained from either Biosera or PAA Laboratories), 50 U ml⁻¹ penicillin, and 50 μg ml⁻¹ streptomycin (Cambrex Corporation). E8.5 to E10.5 embryos were staged by counting somite pairs. 23GFP transgenic embryos were identified by fluorescence illumination (X-Cite 120; Improvision) on a Leica MZFLIII microscope. Mice were housed with free access to food and water. All procedures were in compliance with United Kingdom Home Office regulations and approved by the Ethical Review Committee of the Clinical School and the Home Office.