Design of the mutant Ctip^S326A allele and identification of heterozygous ES cells with the Ctip^S326A-hyg or Ctip^S326A-neo knock-in alleles. The wild-type Ctip locus encompassing exons 9–14 is shown (A), along with the Ctip^S326A-neo targeting vector (B), and maps of the Ctip locus after homologous recombination (C) and cre-mediated recombination (D). For the targeting vector, a neomycin expression cassette flanked by loxP signals (closed triangles) was inserted into the HpaI site of intron 10, whereas the Ctip^S326A mutation (asterisk) and an AgeI restriction site were introduced into exon 11. An HSV thymidine kinase (HSV-TK) gene cassette was included in the targeting vector for negative selection. The wavy line represents plasmid sequences of the targeting vector. Relevant restriction enzyme sites are: PvuII (P), EcoRI (E), HpaI (H), and AgeI (A). The Ctip probe used for Southern analysis and the sizes of the PvuII fragments recognized by the probe are shown. An analogous Ctip^S326A-hyg targeting construct was prepared by replacing the loxP-flanked neomycin resistance cassette in intron 10 with a loxP-flanked hygromycin selection marker (note: the hygromycin cassette lacks a PvuII restriction site). To identify heterozygous ES cells with the Ctip^S326A-hyg or Ctip^S326A-neo knock-in alleles, Southern analysis of PvuII-digested genomic DNA with a 5′ flanking probe (A) was used to screen (E) hygromycin-resistant Ctip^+/− ES cell subclones targeted with the Ctip^S326A-hyg construct and (F) neomycin-resistant 129/Sv ES cell subclones targeted with the Ctip^S326A-neo construct. The 7.9-kb PvuII germline fragment is converted into a 10.4-kb fragment in properly targeted Ctip^{S326A-hyg/−} ES subclones (lanes 2, 4, and 7; panel E) or a 5.8-kb fragment in properly targeted Ctip^S326A-neo/+ ES subclones (lanes 1, 2, and 11; panel F).