The FSCN2 genomic clone was obtained from a 129SVJ mouse library. A fragment lacking a guanine at nucleotide 208 of the mouse FSCN2 gene, corresponding to the human 208delG mutation of FSCN2, was generated by polymerase chain reaction (PCR). Mixtures of 256-bp PCR product with one primer pair (5′-ACAAGACAGAGGGAGGCAGCATTG-3′, p21, and 5′-CTCTCTGCAGTAGGTAGCGGTCGGCAT-3′, p18) and an 186-bp PCR product with the other primer pair (5′-CGACCGCTACCTACTGCAGAGAGCTTTG-3′, p19, and 5′-ATCCATCTCACAGGCCACACGTCCA-3′, p30) were used as templates to create a 419-bp fragment lacking guanine, with the sense primer p21 and the antisense primer p30. The fragment replaced the BstXI/SmaI region (284 bp) on the 5′ genomic flanking sequence (10.5 kb) subcloned into the EcoRI and KpnI sites of a vector (pBlueScript; Stratagene, La Jolla, CA). The floxed pMC1-neo poly(A) fragment, the 3′ genomic flanking sequence (2.3 kb), and the DT-A (diphtheria toxin A-chain) fragment were ligated at the 3′ end of this fragment. An 853-bp fragment was created in which exon 1 was replaced by the cDNA of enhanced green fluorescein protein (EGFP; BD-Clonetech, Palo Alto, CA) in frame, by PCR. The mixture of a 118-bp PCR product obtained with one primer pair (5′-AGATAAACAGATCTGGGCCTCAGG-3′, p17, and 5′-CCCTTGCTCACCATCTTTGAGGCTAGCCACGT-3′, pG2) and a 762-bp PCR product with the other primer pair (5′-GCTAGCCTCAAAGATGGTGAGCAAGGGCGAGGA-3′, pG1, and 5′GGCTGATTATGATCTAGAGTCGCG-3′, pG6) was used as a template to create the 853-bp fragment, obtaining EGFP with the sense primer p17 and the antisense primer pG6. The fragment was replaced with the NheI/KpnI region (1521 bp) on the 3′ genomic flanking sequence (2.7 kb) and ligated DT-A fragment at the 5′ end of this fragment. Finally, we ligated pMC1-neo poly(A) and 5′ genomic flanking sequence (8.8 kb) on the 3′ end of the fragment. Each targeting vector was linearized with the NotI site and electroporated into J1 embryonic stem (ES) cells. Twelve clones of 120 G418-resistant clones that had a point mutation (p-type) and 17 clones of 140 G418-resistant clones in which exon 1 was replaced by EGFP (g-type) underwent homologous recombination, as determined by Southern blot analysis. Two p-type-positive clones and one g-type-positive clone were injected into C57BL/6 blastocysts, resulting in the birth of male chimeric mice. Germline transmission of the disrupted FSCN2 alleles was determined by mating these chimeric mice with C57BL/6 females.