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Review
. 2014 Apr 1;20(10):1655-70.
doi: 10.1089/ars.2013.5293. Epub 2013 Jun 20.

The use of the Cre/loxP system to study oxidative stress in tissue-specific manganese superoxide dismutase knockout models

John C Marecki  1 , Nirmala Parajuli, John P Crow, Lee Ann MacMillan-Crow
Affiliations
Review

The use of the Cre/loxP system to study oxidative stress in tissue-specific manganese superoxide dismutase knockout models

John C Marecki et al. Antioxid Redox Signal. .

Abstract

Significance: Respiring mitochondria are a significant site for reactions involving reactive oxygen and nitrogen species that contribute to irreversible cellular, structural, and functional damage leading to multiple pathological conditions. Manganese superoxide dismutase (MnSOD) is a critical component of the antioxidant system tasked with protecting the oxidant-sensitive mitochondrial compartment from oxidative stress. Since global knockout of MnSOD results in significant cardiac and neuronal damage leading to early postnatal lethality, this approach has limited use for studying the mechanisms of oxidant stress and the development of disease in specific tissues lacking MnSOD. To circumvent this problem, a number of investigators have employed the Cre/loxP system to precisely knockout MnSOD in individual tissues.

Recent advances: Multiple tissue and organ-specific Cre-expressing mice have been generated, which greatly enhance the specificity of MnSOD knockout in tissues and organ systems that were once difficult, if not impossible to study.

Critical issues: Evaluating the contribution of MnSOD deficiency to oxidant-mediated mitochondrial damage requires careful consideration of the promoter system used for creating the tissue-specific knockout animal, in addition to the collection and interpretation of multiple indices of oxidative stress and damage.

Future directions: Expanded use of well-characterized tissue-specific promoter elements and inducible systems to drive the Cre/loxP recombinational events will lead to a spectrum of MnSOD tissue knockout models, and a clearer understanding of the role of MnSOD in preventing mitochondrial dysfunction in human disease.

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Figures

<b>FIG. 1.</b>
FIG. 1.
The mitochondrial electron transport chain is a major site of O2•− production and MnSOD. The electron transport chain is composed of five multimeric protein complexes (Complex I/CI, Complex II/CII, Complex III/CIII, Complex IV/CIV, and ATP synthase) in which, electrons (e−) are sequentially transferred from electron donors (e.g., NADH, FADH2) to electron acceptors culminating in the four-electron reduction of oxygen to water. Protons generated during this process are pumped from the matrix into the intermembrane space forming an electrochemical proton gradient, and are subsequently pumped back into the mitochondrial matrix via ATP synthase to produce ATP. During the electron transfer process, some electrons leak into the matrix (via CI and CIII) and the intermembrane space (via CIII) to form O2•−. MnSOD, residing in the mitochondrial matrix, scavenges O2•− to produce H2O2, which is further detoxified by GPx and Cat. Cat, catalase; GPx, glutathione peroxidase; H2O2, hydrogen peroxide; O2•−, superoxide.
<b>FIG. 2.</b>
FIG. 2.
The Cre/loxP system used to create tissue-specific MnSOD knockout animals. (A) The Cre-expression cassette is cloned downstream of a tissue-selective promoter, often containing additional tissue-specific transcriptional control elements, and is introduced into a mouse genome using standard transgenesis. (B) The tissue-specific Cre-expressing animal is mated with animals engineered with functional MnSOD alleles containing intronic loxP sites flanking exon 3. (C) Cre-mediated recombination leads to the excision of exon 3 from the genome and the loss of a functional MnSOD allele.
<b>FIG. 3.</b>
FIG. 3.
MnSOD knockout in the kidney is limited to Cre-expressing tissues. Mice with the floxed MnSOD gene (left) that are crossed with transgenic mice expressing Cre under the control of the Ksp-Cadherin promoter (middle) produce mice with renal tissue-specific MnSOD deficiency (MnSOD−/−) (right). In the kidneys of the MnSOD−/− mice, Cre-recombinase (indicated by the light circle) is expressed in the distal nephron compartments, including the distal tubules, collecting ducts, and loops of Henle leading to a loss of MnSOD expression (indicated by the white circles). C, cortex; IM, inner medulla; Ksp-cadherin, cadherin 16 promoter; OM, outer medulla.
<b>FIG. 4.</b>
FIG. 4.
The observed phenotypes in the tissue-specific MnSOD knockout models may be broadly categorized by the severity of injury. Based on the characterized phenotypes of the tissue-specific MnSOD knockout models (summarized in Table 1), the animals may be broadly grouped into three categories: (i) no observed or apparent phenotype (e.g., Alb-Cre-mediated liver-specific knockout) in which, the tissue appears unaffected by the loss of MnSOD (although the characterization of oxidative stress and mitochondrial function may have been incomplete), (ii) compensatory phenotype (e.g., Lck-Cre-mediated T cell-specific knockout) in which, the mitochondria are damaged yet other mechanisms may compensate for any loss of function leading to a minimal tissue phenotype, and (iii) massive phenotype with reduced lifespan (e.g., MCK-Cre-mediated heart/muscle-specific knockout) in which, the viability of the organism is severely compromised.
<b>FIG. 5.</b>
FIG. 5.
The phenotypes in animals with tissue-specific MnSOD deficiencies may depend on the balance between the levels of mitochondrial O2•− and compensatory mechanisms within the tissues. In the absence of MnSOD, the level of mitochondrial O2•− may exceed a minimal threshold level such that the organ or tissue must activate cellular mechanisms to heighten resistance to oxidants, to induce rapid endogenous regeneration or increase mitophagy and biogenesis to compensate for oxidative stress and mitochondrial damage. Once the level of O2•− exceeds the endogenous compensatory mechanisms, the mitochondria become severely injured resulting in a major phenotype. The balance between the level of O2•− and the degree to which the tissue is capable of compensating determines the severity of the phenotype, thus it is critical to ascertain the extent of mitochondrial damage as well as mitochondrial repair and regeneration pathways in the tissue-specific MnSOD knockout models.
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References

    1. Agnihotri N, Kaur S, Dilawari JB, Bhusnurmath SR, and Kaur U. Diminution in parietal cell number in experimental portal hypertensive gastropathy. Dig Dis Sci 42: 431–439, 1997 - PubMed
    1. Andersen JK. Oxidative stress in neurodegeneration: cause or consequence? Nat Med 10Suppl: S18–S25, 2004 - PubMed
    1. Bakthavatchalu V, Dey S, Xu Y, Noel T, Jungsuwadee P, Holley AK, Dhar SK, Batinic-Haberle I, and St. Clair DK. Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Polgamma against UV-induced inactivation. Oncogene 31: 2129–2139, 2012 - PMC - PubMed
    1. Belteki G, Haigh J, Kabacs N, Haigh K, Sison K, Costantini F, Whitsett J, Quaggin SE, and Nagy A. Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction. Nucleic Acids Res 33: e51, 2005 - PMC - PubMed
    1. Birling MC, Gofflot F, and Warot X. Site-specific recombinases for manipulation of the mouse genome. Methods Mol Biol 561: 245–263, 2009 - PubMed

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