Fig. 2
Generation of neuron-specific Slc38a1 knockout mice. a Targeting strategy to create the floxed Slc38a1 allele (Slc38a1flox). Slc38a1 exon 2 is flanked by loxP sites. The flippase recombinase target-flanked Neo cassette was removed by crossing with CAG-FLP mice. Exon 2 was removed by crossing with SynI-Cre mice to selectively produce the Δ allele in neurons. b Southern blot analysis to confirm the recombination with the targeting vector at the genomic Slc38a1 locus. Genomic DNA from embryonic stem cells was digested with AfIII and hybridized with a DIG-labeled 3′ probe. c PCR analysis verifying the Δ allele in mutant mice. Genomic DNA was extracted from the brain of each indicated mouse, and PCR products derived from the wild-type, flox, or Δ allele were detected. d Quantification of Slc38a1 and Slc38a2 mRNA levels in whole brains of from mutant mice. Total RNAs were extracted from whole brains of control or mutant mice, and the mRNA levels of Slc38a1 and Slc38a2 were compared using qRT-PCR. Values were normalized to those of Gapdh (n = 3). e Deletion efficiency of Slc38a1 in brain segments. Proteins were extracted from each indicated brain segment of control or mutant mice, and SNAT1 was detected using western blotting. CBB staining was used as a loading control. C and M indicate control and mutant, respectively. f Confirmation of neuron-specific Slc38a1 deficiency in mutant mice. Double-immunohistochemical staining using antibodies against SNAT1 and NeuN. Nuclei were counterstained with Hoechst 33342. Scale bars indicate 100 µm
