The MnSODtet-on targeting vector was designed by Drs. J. Richard Chaillet, Joel S. Greenberger and Michael W. Epperly (University of Pittsburgh School of Medicine) to insert a 5.3 kbp Tet-On regulatory cassette approximately 30 nucleotides upstream of the initiation codon in the first exon of the manganese superoxide dismutase (Sod2 or MnSOD) gene on chromosome 17. The Tet-On regulatory cassette contains the optimized form of reverse tetracycline controlled transactivator (rtTA-M2), polyA sequence, loxP site, neomycin resistance gene (neor), polyA sequence, loxP site and five copies of tetracycline operator 5prime of a minimal cytomegalovirus promoter (tetO+CMV [or teto5 CMV]). The Tet-On regulatory fragment is a modification of the neor version of Tet-Off regulatory cassette. The Tet-Off cassette was converted to Tet-On by changing five codons via site-directed mutagenesis: S12G(GGC), E19G(GGG), A56P(CCC), D148E(GAG) and H179R(CGC). These amino acid changes converted tTA to the M2 form of rtTA (rtTA-M2). The MnSODtet-on construct was electroporated into 129S6/SvEvTac-derived TC1 embryonic stem (ES) cells. Chimeric animals were bred with C57BL/6NTac mice to establish the colony. The MnSODtet colony was backcrossed onto the C57BL/6NTac genetic background for six generations. In 2014, black heterozygous males were sent to The Jackson Laboratory Repository. Upon arrival, males were used to cryopreserve sperm. To establish our living MnSODtet mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).
