Targeted disruption of the mouse Atp1a2. Targeted disruption of the mouse Atp1a2. A, Targeting strategy for mutating the Na+, K+-ATPase α2 subunit gene (Atp1a2). Exons are represented as black boxes. A neomycin-resistant gene cassette (neo, depicted as a white box) was inserted in the opposite orientation into exon 2, and a bacterial diphtheria toxin subunit gene (DTA, depicted as a gray box) was inserted for negative selection. The targeted allele was verified by PCR (data not shown) and Southern blot analyses with the indicated probe in A. B, Southern blot analysis of genomic DNA. The XhoI DNA fragments from the wild-type (∼17 kb) and targeted (5 kb) alleles are shown. C, RT-PCR analysis of total RNA isolated from the E18.5 brain of each genotype. Positions of PCR products for the α2 subunit and β-actin as a control are indicated. Note that the α2 subunit PCR product is absent in Atp1a2-/- mice (-/-). D, A microsome fraction was prepared from the brains of E18.5 mice, and 40 μg of protein was analyzed by Western blotting with an anti-α1 (1:2000), an anti-α2 (1:1000), or an anti-α3 (1:2000) antibody. Note that the α2 subunit polypeptide is absent in Atp1a2-/- mice. Keiko Ikeda et al. J. Neurosci. 2004;24:10693-10701 ©2004 by Society for Neuroscience