B6N.129S6-Sod2^{tm1(rtTA*M2,tetO)Jsg}/J

Stock number: 024851
Product name: MnSOD^tet
Research areas: Cell Biology Research, Metabolism Research, Neurobiology Research, Research Tools

Description

MnSOD^tet functions as a knockout allele (abolishes MnSOD expression) in the absence of doxycycline. MnSOD^tet mice may be useful for studying the role of antioxidants in cellular metabolism and aging, and in mitochondria function.

Detailed description

MnSOD (Sod2) is a major antioxidant for the conversion of superoxide produced in the mitochondria during respiration. It has also been shown to be important in protecting the mtiochondria and the cell from irradiation damage. These mice may be useful for studying the role of antioxidants in cellular metabolism aging and in the functioning of the mitochondria.

MnSOD^tet functions as a knockout allele (abolishes MnSOD expression) in the absence of doxycycline (dox). Both male and female heterozygous (MnSOD^tet/+) mice are viable and fertile with normal phenotype, regardless of doxycycline treatment. The donating investigator reports that in the absence of dox, homozygous (MnSOD^tet/tet) newborn mice die within seven days of birth, similar to MnSOD knockout mice. Homozygous mice are smaller than heterozygous or wildtype littermates until weaning, and are similar sized thereafter. At around 5 to 6 weeks of age, homozygotes can be taken off dox and are able to maintain body weight and health for at least one month. The donating investigator also reports that when breeding heterozygotes together, 1 of 10 homozygous pups given dox will survive to weaning. Administration of dox water to nursing dams does not lead to increased survival of homozygous pups; the most effective way to keep homozygous pups alive is by dox injection. Homozygous mice on dox are fertile but have fewer litters.

Development

The MnSOD^tet-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 (neo^r), polyA sequence, loxP site and five copies of tetracycline operator 5prime of a minimal cytomegalovirus promoter (tetO+CMV [or tet_o5 CMV]). The Tet-On regulatory fragment is a modification of the neo^r 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 MnSOD^tet-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 MnSOD^tet 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 MnSOD^tet mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ inbred females (Stock No. 005304).

Allele

Symbol: Sod2^{tm1(rtTA*M2,tetO)Jsg}
Name: targeted mutation 1, Joel Greenberger
MGI accession ID: MGI:5568594
Generation method: Targeted
Attributes: Inducible; Null/Knockout; Inserted expressed sequence
Marker symbol: Sod2
Marker name: superoxide dismutase 2, mitochondrial
Chromosome: 17
Strain of origin: 129S6/SvEvTac
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli
Site of expression: Administration of doxycycline controls expression of Sod2 in mitochondria.
Molecular note: The MnSOD targeting vector was designed to insert a 5.3 kbp Tet-On regulatory cassette approximately 30 nucleotides upstream of the initiation codon in the first exon. The Tet-On regulatory cassette contains the optimized form of reverse tetracycline controlled transactivator (rtTA-M2), polyA sequence, loxP site, neomycin resistance gene (neo), polyA sequence, loxP site and five copies of tetracycline operator 5' of a minimal cytomegalovirus promoter. The Tet-On regulatory fragment is a modification of the neo 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).