Figure 1. Targeted Disruption of the BDNF Locus.

(A) Schematic showing the portion of the BDNF prohormone that is deleted and replaced by the phosphoglycerate kinase (PGK)–neo selectable marker in the BDNPO^neo mutant. The signal sequence (SS), pro, and mature regions are indicated by closed, hatched, and closed bars, respectively. The two arrowheads indicate the limits of the deletion. The Cs and lines indicate the positions of cysteine residues and disulfide bonds in the mature portion of BDNF, as predicted from the structure of NGF (McDonald et al., 1991).

(B) Schematic showing the strategy used to disrupt the BDNF gene. The thin horizontal lines represent murine genomic DNA. The exon encoding BDNF is indicated by a box. The portion of the exon replaced by the PGK–neo cassette (arrow indicates direction of transcription) is indicated by broken lines. The targeting construct is shown as linearized for transfection with the thymidine kinase gene used for negative selection (open box labeled MCITK; arrow indicates direction of transcription) and pBluescript KS vector (open box labeled pBSKS). The probe used for Southern blot analysis is indicated by the closed bar. Arrows above the schematic of the mutated BDNF gene show the approximate positions of oligonucleotide primers used for polymerase chain reaction. Restriction site abbreviations: R, EcoRl; Sa, Sacl; Ap. Apal; Bg, Bglll; S, Sall.

(C) Southern blot analysis. The probe was derived from sequences outside the targeting construct (see [B]). A single restriction fragment of the size expected to result from introduction of an EcoRl restriction site into the BDNF locus by homologous recombination with the targeting construct is detected in mice homozygous for both mutant alleles (1.7 kb in mutants, 15.5 kb in wild type). EcoRl digested genomic DNA samples from mice homozygous for BDNP^neo1 (lanes 1 and 2) wild type (lanes 3 and 4) and BDNF^neo2 (lanes 5 and 6).