A MusD Retrotransposon Insertion in the Mouse Slc6a5 Gene Causes Alterations in Neuromuscular Junction Maturation and Behavioral Phenotypes
Figure 2
Characterization of the new Slc6a5 mutation as a null-allele.
A. Schematic of the Slc6a5 gene organization, with boxes and lines representing exons and introns respectively, and boundaries of PCRs (1 to 4) run on reverse-transcribed cDNAs. B. Agarose gel of the PCR amplification products from cDNA. Note the extra band in PCR 2 on mutant brain cDNAs (arrow). C. Schematic representation of intron 5, and location of the insertion and splice donor and acceptor sites in the retrotransposon sequence. The 10 base pairs immediately 5′ of the insertion site, 1833 bp into intron 5, are shown, as well as the first base-pairs of the LTR element of the retrotransposon. The 183 bp sequence of the transposon that is spliced-in Slc6a5 mRNA is indicated with its 10 first and last base-pairs and the inferred acceptor and donor splice sites. The primers used to localize the insertion (int5F and MusR), and to amplify the transposon from mutant genomic DNA (int5F and int5R) are indicated by arrows. D. Agarose gel of the products of the PCR on genomic DNA with primers flanking the insertion site (int5F/R of C.). A 3 kb product was amplified from wild-type gDNA, but only a 9 kb product was amplified from mutant gDNA. E. Anti-GLYT2 Western blot on spinal cord cell membrane preparations, with beta-dystroglycan used as a loading control. Genotypes are indicated above the blot. F. Anti-GLYT2 immunocytochemistry (DAB) on brain sagittal sections and spinal cord cross sections. Nuclei were counterstained with hematoxylin before mounting. The lateral brain stem is shown in the top panels with the ventral lobules of the cerebellum visible in the top right corners and the ventral horn of the spinal cord in shown in the bottom panels.