In utero Jag1 overexpression in Fzd3 mutants

The Jag1 coding sequence was recovered from plasmid Addgene 17336 (ref. 58 (link)) encoding the rat Jag1 (mouse and rat Jag1 proteins are 99% identical), and cloned in the Cdk5r vector (kindly provided by Dr Paola Arlotta). The construct was verified by sequencing. The plasmid was electroporated together with a green fluorescent protein (GFP) encoding plasmid. In utero microinjection and electroporation was performed at E13.5, using pregnant Fzd3^+/− females; mutant embryos were identified by their looptail phenotype. Foetuses were examined at E18.5 and genotypes were confirmed using tail clips. Needles for microinjection were pulled from Wiretrol II glass capillaries (Drummond Scientific) and calibrated for 1 μl injections. DNA solutions were mixed in 10 mM Tris, pH 8.0, with 0.01% Fast Green. Forceps-type electrodes (Nepagene) with 5 mm pads were used for electroporation (five 50-ms pulses of 30 V)59 (link).