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Fig. 1. Targeted mutagenesis of the Gucy2d, Sema3f and Sema3b loci. (A) Genomic structure of the Gucy2d locus on chromosome 7E1 according to the NCBI database. The coding region is spread over 19 exons, with at least another three exons at the 5' untranslated region. No other genes are predicted within the Gucy2d locus. (B) GC-D knock-in mutations. A PacI site (P) was created 3 bp downstream of the stop codon in exon 22 by PCR subcloning between NcoI (N) and SacI (S). The targeting vector encompasses the region from an upstream EcoRI (R) site to a downstream XhoI (X) site. Left, targeting vector. Right upper, targeted mutation in ES cells. Right lower, targeted mutation after germline transmission. Diagram is not to scale. (C) Genomic organization of the Sema3f and Sema3b loci on chromosome 9F2. The coding regions comprise 17 exons spanning 22 kb and 6 kb, respectively. At least three other genes (Gnai2, Slc38a3 and Gnat1) are predicted within the 80 kb between Sema3f and Sema3b. (D) Targeting strategies to create knockout mutations of Sema3f (left) and Sema3b (right). Gene targeting replaces exons 2-15 (Sema3f) or exons 1-17 (Sema3b) with the ACNF cassette. During germline transmission in male chimeras, the ACNF cassette excises itself, removing most (Sema3f) or all (Sema3b) of the ORF. Diagrams are not to scale.

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