Fig. 1. Generation of conditional Robo1 knockout mice. (A) Exon structure (depicted by a square box) corresponding to Ig domains 2b, 3a and 3b (exons 4 to 6, respectively, indicated in red). The position of the terminal nucleotides within an amino acid codon is indicated above the exon in red. The yellow triangles shown in the upper panel represent lox P sites cloned into unique XbaI and KpnI sites (loxP sites flanking a neo cassette, not shown in diagram). (B,C) Genotype analysis of tail DNA obtained from one F2 litter. Allele sizes are discriminated by the XbaI site that was `silenced' by the cloning of the loxP site between exons 4 and 5 and probed with a genomic probe including exon 4 (B) or by PCR across exons 4 and 6 (C). (D) RT-PCR across exons 3 and 7 on mRNA isolated from wild-type and homozygous brains also confirmed that the deletion had occurred in the mutants. (E) The mRNA sequence of the RT-PCR products that were cloned into a TA vector. The lower panel (mutant) indicates the presence of multiple premature stop sites within the protein. (F) In situ hybridization was performed using probes against Robo1, Robo2 and Robo3. Robo1 mRNA was absent, but Robo2 and Robo3 mRNA were still expressed in the mutant. (G) Similarly, Robo1 protein was not expressed in the mutant as indicated by western blot analysis.