A strategy for the ubiquitous overexpression of human catalase and CuZn superoxide dismutase genes in transgenic mice

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Abstract

In the present study, we generated transgenic mice that overexpress catalase or CuZn superoxide dismutase (CuZnSOD) in all tissues using large genomic DNA fragments. An 80 kb human genomic DNA, containing the 33 kb human CAT gene as well as the 41 kb of 5′ and the 6 kb of 3′ flanking regions, was obtained by screening a human P1 library and was used to produce transgenic mice Tg(CAT). Transgenic mice Tg(SOD1) were produced by a similar strategy using a 64 kb human genomic DNA containing the 10 kb human SOD1 gene and the 27 kb of both 5′ and 3′ flanking regions. Catalase mRNA levels were 2–6- fold higher and catalase activity levels were 2–4- fold higher in the various tissues of the hemizygous Tg(CAT) mice compared with wild type mice. The mRNA levels for CuZnSOD were 2–12- fold higher and the CuZnSOD activity levels were 2–5- fold higher in the hemizygous Tg(SOD1) mice compared with wild type mice. In summary, our study demonstrates that a strategy of using large genomic DNA containing either the entire human CAT or SOD1 gene with large flanking regions gives ubiquitous increased expression of CuZnSOD and catalase. In addition, the expression of catalase closely reflects the tissue specific pattern found in the endogenous gene. These transgenic mice will be useful in studying the role of oxidative stress/damage in aging and age-related pathologies.

Introduction

One of the difficulties in aging research is the ability to determine whether a change in a specific gene or physiological process plays a role in the mechanism of aging. Therefore, investigators have been forced to correlate changes in a biochemical process or a gene with the age of an organism. While these types of experiments provide investigators with information that can be used to support or refute various theories of aging, they do not allow one to test directly the role of a specific gene in aging. With the ability to genetically engineer animals, investigators now have experimental systems whereby a specific gene or process can be altered, and by which the effect of this alteration on aging can be studied.

Although transgenic animal models are potentially powerful tools for studying aging, one of the problems in generating transgenic models for aging studies is the selection of the enhancer/promoter to drive the expression of the transgene. Due to the global nature of the aging process (i.e. aging potentially affects all cells and no one tissue appears to be responsible for aging), transgenic experiments designed to test many possible mechanisms of aging require that the gene or process of interest be altered in most, if not all, tissues of the transgenic animals in which the gene is expressed. For example, to test oxidative stress theory of aging, it is necessary to develop transgenic animals that show altered resistance to oxidative stress in all tissues of the organism.

To achieve the global expression of a transgene, investigators have used the promoters of ubiquitously expressed genes to drive the expression of genes of interest (Richardson et al., 1997). Unfortunately, most of the promoters and enhancers that have been characterized in transgenic mice direct the expression of the transgene to one or a few tissues (Richardson et al., 1997, Morgan et al., 1999). Moreover, when using such constructs, overexpression is often very high and can vary widely between founder animals and among tissues within one animal (Palmiter and Brinster, 1986). Therefore, these transgenic models are of limited usefulness in modeling gene overexpression in a controlled manner to study its effect on the aging of whole animals. An alternate approach is to use large genomic DNA fragments containing the gene (introns and exons) of interest and the 5′- and 3′- flanking regions, which contain the regulatory sequences necessary to drive the proper expression of the transgene. The cloning of genomic DNA fragments that are sufficiently large enough to span an entire gene became possible with the introduction of P1 bacteria phage vector in the early 1990s. The P1 vector can package genomic DNA fragments up to 100 kb in length (Sternberg, 1990, Sternberg, 1992). P1 clones have been used to generate transgenic mice successfully in several laboratories (Linton et al., 1993, Callow et al., 1994, Yan et al., 1998a, Yan et al., 1998b). Using a 45 kb human genomic fragment that contained the 12 kb human renin gene as well as 25 kb of the 5′-flanking region and 8 kb of the 3′-flanking region, Yan et al. (1998a) produced transgenic mice that express the renin transgene in a similar expression pattern as the endogenous renin gene, e.g. only in kidney, adrenal, lung, eye, ovary, and brain. In contrast, when the transgenic mice were generated using a 13 kb genomic DNA fragment, which contained only 0.9 kb of the 5′-flanking region of the gene, the human renin transgene displayed a much broader tissue distribution, including many tissues not normally associated with renin gene expression, e.g. adipose tissue, muscle, spleen, heart, etc. (Sigmund et al., 1992, Yan et al., 1998a). These studies show that endogenous gene expression pattern of a transgene can be achieved using a genomic DNA fragment containing large flanking sequences.

Currently, one of the major theories of aging is the free radical, or oxidative stress, theory of aging. The strongest evidence for this theory comes from studies using Drosophila. Transgenically altered Drosophila, which have increased expression of CuZn superoxide dismutase (CuZnSOD) alone or in combination with catalase, have been reported to have a significantly longer life span (Orr and Sohal, 1993, Orr and Sohal, 1994, Parkes et al., 1998, Sun and Tower, 1999, Phillips et al., 2000). Although the Drosophila studies strongly suggest a role for oxidative stress/damage in aging, it is critical that these experiments be replicated in mammals. In this study, we show that transgenic mice can be generated using large genomic fragments that contain either the human CAT gene or the human SOD1 gene and that these transgenic mice show ubiquitous overexpression of catalase or CuZnSOD. These animals will be valuable models for testing the free radical theory of aging.

Section snippets

Isolation and characterization of P1 clones

We isolated large fragments of human genomic DNA that contain either the CAT or SOD1 gene by screening a human P1 genomic library (Genome Systems, Inc., St. Louis, MO). The primer pair (forward 5′- tcc gag ccc att ggg ctt cc-3′ and reverse 5′- atc ccg gct gtc agc cat ag-3′) that amplifies a PCR product of 219 bp from exon 1 of the human CAT gene was used for the screening. One P1 clone containing the human CAT gene, designated PCAT, was identified. A P1 clone (PSOD1) containing the human SOD1

Results

We used large fragments of genomic DNA containing either the entire human CAT or SOD1 gene (introns and exons) and large segments of flanking regions to generate transgenic mice that overexpress the antioxidant enzymes catalase and CuZnSOD. The purpose of using this approach was to maximize the chance of having the enhancer/promoter elements necessary for expressing these antioxidant genes in a tissue-specific manner that mimics the expression of the endogenous genes.

Transgenic mice Tg(CAT)

Discussion

The free radical, or oxidative stress, theory of aging states that progressive and irreversible accumulation of oxidative damage with age, due to an imbalance between pro-oxidants and antioxidants, results in age-related loss of physiological functions. Strong evidence supporting this theory has come from studies in invertebrates using selected long-lived mutants or transgenic animals. For example, the age-1 (daf-2 gene) mutants of Caenorhabditis elegans, which have increased longevity, have

Acknowledgements

This work was supported by a Merit grant from the Department of Veteran Affairs, Program Project Grants PO1AG14674 and PO1AG17242 the Nathan Shock Center of Excellence in Basic Biology of Aging (PO3 AG13319) at the University of Texas Health Science Center at San Antonio. We would like to thank the transgenic core facility of the Beth Israel Deaconess Medical Center (Boston, MA) for producing the transgenic mice.

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    1

    These people contributed equally to the work.

    2

    Present address: Genome Therapeutics Corporation, 100 Beaver Street, Waltham, MA 02453, USA.

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