Figure 2. Targeting strategy and confirmation of knockdown.

A) Cartoon indicating the targeting strategy. Disruption of the Vangl2 gene was achieved by modification of the wild type allele to insert LoxP sites flanking exon 4. Expression of Cre recombinase results in the excision of exon 4 and subsequent loss of the transmembrane domains. B) RT-PCR on RNA isolated from whole E10.5 Vangl2^flox/flox; Sox2-Cre embryos showed that there was no Vangl2 transcript produced in the mutants, although this was abundant in controls. Actin was used as a loading control. C) Western blotting using protein isolated from whole E15.5 Vangl2^flox/flox; Sox2-Cre embryos showed that there was a major reduction in Vangl2 protein in the mutant embryos, although the presence of a faint band suggested that the Cre was not 100% efficient at later stages. Gapdh was used as a loading control. D–K) Immunohistochemistry for Sox2-Cre (using eYFP as a reporter for Cre expression) showed that recombination was variable across the embryo in the mutants (E,I). However, immuno-staining for Vangl2 showed that the protein was lost from the outflow tract (J, compare to F; in F strong staining is apparent within the OFT and neural tube - arrows). Also see S2 Fig. OFT - outflow tract, Vangl2^f – Vangl2^flox. Scale bar  = 200 µm.