[34 Manganese Superoxide Dismutase Transgenic Mice Characteristics and Implications

By Ching K. Chow, Hsiu-Chuan Yen, Wissam Ibrahim, and Daret K. St. Clair


Aerobic organisms are protected from oxidative stress by a variety of interacting antioxidant systems under normal conditions. Among the antioxidant systems, superoxide dismutase (SOD) is considered the first line of defense against oxidative stress.1,2 There are two types of intracellular SOD: the manganese-containing SOD (MnSOD) and the copper-zinc-containing SOD (Cu,ZnSOD), and one extracellular SOD (EC-SOD), mainly the copper-zinc form, in mammalian organs. By rapidly removing superoxide, SOD reduces the tissue concentration of superoxide and prevents the production of reactive hydroxyl radical and peroxynitrite. MnSOD is a critical antioxidant enzyme in aerobic organisms because the superoxide is mainly generated on the matrix side of the inner mitochondrial membrane where MnSOD is located.3 The essentiality of MnSOD is evidenced by the findings that deficiency in superoxide dismutases, but not catalase, shortens the life span of yeast cells4; that MnSOD [but not Cu,ZnSOD, catalase, or glutathione (GSH) peroxidase] knockout mice die within the first few weeks of life5,6; that overexpression of Cu,ZnSOD does not prevent neonatal lethality in mutant mice that lack MnSOD7; and many other reports.

As a large variety of antioxidant systems are involved in protecting aerobic organs against oxidative stress, the contribution of an individual antioxidant system to the overall antioxidant defense in intact cell/animals is difficult to assess. The application of genetic manipulation to create cells/animals that are specifically enriched with or lack one or more of the antioxidant enzymes represents a physiological avenue to understand their specific roles in situ. A number of genetically altered mice with increased or decreased expression of important antioxidant

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enzymes, MnSOD,5'6'8-11 Cu,ZnSOD,12"14 EC-SOD,15"17 catalase,18-19 and/or GSH peroxidase,20'21 have been produced. The use of these transgenic mice and mutant to study free radical-induced oxidative damage has provided a better understanding of the role of individual antioxidant enzymes in antioxidant defense. However, over- or underexpression of a single antioxidant enzyme may shift the balance of cellular redox status and/or result in compensatory changes in other antioxidant systems. We describe the procedures employed to identify and characterize MnSOD transgenic mice used in our laboratories.

Identification and Characterization of MnSOD in Transgenic Mice

The MnSOD transgenic mice used in our laboratories were prepared from the Fi progeny of C57BL/6x C3H hybrids (B6C3) at the transgenic animal facility of the University of Kentucky (Lexington, KY), using standard procedures.22 Human MnSOD cDNA under the transcriptional control of the human /¡-actin promoter as used for the generation of the human MnSOD transgene. The DNA was introduced into pronuclei of fertilized mouse eggs by microinjection. Mice with stable integrated human MnSOD transgenes identified by Southern analysis were selected as founders, and were propagated as heterozygous transgenic mice. Six to 8 weeks after birth, the line of transgenic mice expressing human MnSOD was bred with nontransgenic mice to produce transgenic and nontransgenic offspring. Two

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weeks after birth, the presence of introduced human MnSOD gene in the pups was identified by Southern blot analysis, and expression of steady state mRNA from the human MnSOD gene was confirmed by Northern analysis. Translation of human MnSOD mRNA into protein was identified by Western blotting, and immunogold staining was used to determine the intracellular location of the MnSOD protein in the transgenic mice. Human MnSOD activity was verified by native activity gels, which permit the separation of MnSOD isozymes. The activities of SOD, glutathione (GSH) peroxidase, and catalase, as well as the levels of low molecular weight antioxidants, GSH, ascorbic acid, and vitamin E (principally a-tocopherol), in tissues were assessed to determine whether altered expression of MnSOD results in compensatory or adaptive changes in other important antioxidant systems.

Identification of Human MnSOD Transgene

By using a probe that yields distinct bands for both the foreign DNA and an endogenous gene, Southern blot analysis allows for specific detection of the human MnSOD transgene. After isolation from approximately 0.5-cm mouse tail biopsies, genomic DNA is digested in 0.5 ml of 1 MTris-HCl (pH 8.5), 500 mM EDTA, 200 mA/NaCl, 0.2% (w/v) sodium dodecyl sulfate (SDS), and proteinase K (100 //g/ml) at 55° overnight, and is centrifuged at 13,000g for 10 min at room temperature. The supernatant is then mixed with 0.5 ml of isopropanol and centrifuged at 13,000g for 5 min. The precipitated DNA is then washed twice with 0.5 ml of 95% (v/v) ethanol, and allowed to dry at room temperature. The resulting genomic DNA is redissolved in 100 /xl of 10 mM Tris-HCl and 1 mM EDTA, pH 7.4, and stored at -20°.

Genomic DNA (6-8 /xl) isolated from mouse tail is digested with restriction enzyme Pstl at 37° overnight. The digested DNA is then electrophoresed on a 0.8% (w/v) agarose gel. The gels are then denatured, neutralized, and transferred to a nitrocellulose membrane (Nytran paper; Schleicher & Schuell, Keene, NH). The membrane is air dried, baked at 80° for 2 hr in a vacuum oven, and prehybridized for 5-10 hr at 42° in hybridization buffer [5x saline-sodium phosphate-EDTA buffer (SSPE), 50% (v/v) formamide, 5x Denhardt's solution, 0.1% (w/v) SDS, and denatured salmon sperm DNA (0.1 mg/ml)]. After hybridization with the [32P]dCTP-labeled human MnSOD cDNA probe for 24-48 hr at 42° in hybridization buffer, the membrane is washed twice with membrane wash solution [5x saline-sodium citrate buffer (SSC), 0.1% (w/v) SDS, and 0.05% (w/v) sodium pyrophosphate] for 15 min at room temperature and once with 0.1 x SSC buffer, 0.1% (w/v) SDS, and 0.05% (w/v) sodium pyrophosphate at 65°. The membrane is then air dried and exposed to Kodak (Rochester, NY) XAR film at —80°. Figure 1 shows a Southern analysis of the human MnSOD gene isolated from tails of our transgenic founder mice. The copy number of the human MnSOD gene in each founder mouse varies significantly.


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