Fig. 1. Gene replacement of the BDNF coding sequence with NT3. (A) Schematic diagram of the approach to change the BDNF loci to NT3. The BDNF exon V (shaded box) was opened with BglII (B) and the NT3 coding region was fused in-frame followed by a neomycin cassette. (B) ES cells were analysed for homologous recombination using an external probe for Southern blot analysis (Bs; BstXI and E; EcoRV). The wild-type (WT) allele revealed a 12 kb band while the mutant band was 5.6 kb. (C) Animals were mated with deleter-cre mice to lox out the neomycin cassette. (D-E) Two PCRs were performed to distinguish between animals containing or not containing the neomycin cassette. The PCR primers used for this analysis are depicted in (C). (D) The first PCR primers (Neo-3', Neo-5') are designed to amplify a fragment within the neomycin cassette of about 600 bp. The neomycin fragment is amplified in BDNF^NT3/NT3 mice while no fragment is amplified in mice crossed with deleter-cre mice (BDNF^{NT3lox/NT3lox} mice). (E) In the BDNF^{NT3lox/NT3lox} mice a fragment of about 600 bp is instead amplified using the NT3-04 and BL2 primers, this band is not amplified in animals still carrying the neomycin cassette.