Figure 1
Generation of VGAT-/- mice. (A) Schematic representation of the wild-type VGAT allele, the targeting vector, the VGAT-floxneo allele, and the VGAT knockout allele. Exons are represented by numbered white boxes. LoxP sites (open diamonds) and a PGK-Neo cassette (neo; gray box) flanked by the frt sites (open ellipses) were introduced into the wild-type VGAT locus by homologous recombination to produce the floxneo allele. The probes used for Southern blot analysis are indicated as white (5' probe) and red (3' probe) boxes. The expected sizes of the KpnI- and NcoI-digested genomic DNA fragments hybridized with the 5' and 3' probes, respectively, are indicated as lines under the schemes. Relevant restriction sites are indicated as follows: K, KpnI; N, NcoI. PCR primers are indicated as arrows. (B) (Left) Southern blot analysis of KpnI-digested genomic DNA isolated from VGAT+/+ (+/+) and VGATfloxneo/+ (Floxneo/+) mice using the 5' probe indicated in A. The wild-type allele corresponds to the 15.2 kb band, whereas the floxneo allele corresponds to the 12.0 kb band. (Right) Southern blot analysis of NcoI-digested genomic DNA isolated from VGAT+/+ (+/+), VGAT+/- (+/-), VGAT-/- (-/-), and VGATfloxneo/+ (Floxneo/+) mice using the 3' probe indicated in A. The wild-type allele, the knockout allele, and the floxneo allele correspond to the 10.7 kb, 7.4 kb, and 6.3 kb bands, respectively. (C) Genotyping of offspring from intercrosses of VGAT+/- mice by PCR. Three primers were used (see Methods). Primers P2 and P3 produce a 183 bp fragment that represents the wild-type allele, whereas primers P1 and P3 produce a 430 bp fragment that represents the knockout allele. (D) Western blot analysis of E18.5 whole brain homogenates from VGAT+/+ (+/+), VGAT+/- (+/-), VGAT-/- (-/-) using the anti-VGAT antibody directed against an N-terminal epitope. (E) Ventral views of the upper jaw of E18.5 VGAT+/+ (+/+) and VGAT-/- (-/-) mice. In contrast to the completely fused palate of a VGAT+/+ mouse, secondary palatal shelves of a VGAT-/- mouse did not contact each other (arrows), and its nasal cavity (asterisk) could be seen. (F) Lateral views of E18.5 VGAT+/+ (+/+) and VGAT-/- (-/-) mice. An arrow indicates omphalocele in a VGAT-/- mouse. In addition, the VGAT-/- mouse showed an extremely hunched position in contrast to the VGAT+/+ mouse.