FIG. 1.

Generation and genotyping of MnSOD^tet allele. Panel A: Schematic of mutagenesis approach to generate tetracycline-regulated MnSOD allele. The top line is endogenous MnSOD allele, comprised of five exons (filled rectangles). The middle line is linearized targeting plasmid with Tet-On regulatory cassette inserted in exon 1 approximately 30 nucleotides 5′ of initiation codon. rtTA is coding sequence of reverse tetracycline repressor protein, neo^R is G418 selectable marker gene, and tetO+CMV is comprised of five copies of tetracycline operator 5′ of minimal CMV promoter. Homologous recombination between MnSOD allele and targeting plasmid in ES cells resulted in mutant MnSOD^tet allele (bottom line). Panel B: Southern blot of EcoRI-digested DNA from tails of offspring of cross between heterozygous MnSOD^tet/+ parents. Blot was hybridized with Probe A located 5′ of the MnSOD locus and not present in the targeting plasmid. Panel C: PCR genotyping assay to distinguish wild-type MnSOD and MnSOD^tet alleles. 473-bp PCR product from MnSODwtF and MnSODwtR primers corresponds to wild-type MnSOD allele. 281-bp PCR product from MnSODwtF and MnSODTetR primers corresponds to MnSOD^tet allele.