The genomic fragment 2B13 was digested with EcoRI+NcoI and the resulting piece containing the TATA box, transcription Niflumic acid initiation site and exon 1 was cloned to pBSIIKS vector to give the left arm. Digestion of 2B13 with SpeI+XhoI and cloning the resulting piece containing Isoforskolin intron 1 into pWH9 vector gave the right arm. The pWH9 vector contains the neomycin cassette. The cassette and the right arm were digested with XhoI and cloned to the unique XhoI site in the exon 1. The resulting targeting construct, containing a 5 kb left arm and 2.4 kb right arm was linearized with NotI digestion and electroporated into 129sv embryonic stem cells as described earlier. The ES cells were subjected to G418 selection to obtain stable transfectants. To identify the clones with homologous recombination, EcoRI digested DNA was analyzed by Southern blotting using a 750 bp external probe and 800 bp internal probe. Internal probe identified a fragment in the wild type and fragment in the mutant alleles and nothing in the wild type. Three correctly targeted clones were injected into C57BL/6 blastocysts and the injected blastocysts were transferred into pseudo pregnant foster mothers to generate chimeric mice. Resulting chimeric mice were mated with C57BL/6 mice and the germ line transmission of the mutant allele was tested by Southern blot analysis with the external and internal probes. For routine genotyping PCR reactions were designed with one pair of primers giving a product from the intron 1 and one primer pair with the sense primer in the neomycin cassette and the antisense primer in the intron 1. During the development of the cerebral cortex, neurons are generated in the ventricular zone and then migrate outward to the cortical plate, an event that is called radial migration. These neurons stop their movement and settle into the six layers of the cortex in an inside-out pattern, whereby early-born neurons are positioned in the deeper layers, and later-born neurons are located in the more superficial layers, migrating outward by passing the earlier born neurons. In the later stage of the cortical development, just after radial migration ends, neurons that have settled in the cortex continue to elongate their axons toward their targets.