11 kb i

Fig. 2. Targeted disruption of the mouse Sodl gene. Schematic diagram showing the genomic structure and partial restriction map of the mouse Sodl locus (top), the targeting vector (middle), and the predicted structure of the targeted locus (bottom). Numbered solid boxes represent exons. The hatched box represents the 5' external sequence used as a hybridization probe. B, Bam HI; E, EcoRI; H, WmdIII; P, Pst\; S, Sad: Sa, Sail: Sm, Smal. neo, Neomycin resistance gene cassette; tk, herpesvirus thymidine kinase gene cassette. The predicted sizes of hybridizing Pstl genomic fragments of the wild-type allele and the targeted allele are indicated at the top and bottom, respectively.

of Cu,ZnSOD (SODI) in ischemic heart disease, we have created SOD1 knockout mice by disrupting the Sodl gene.

Targeted Disruption of Mouse Sodl Gene

Mouse Sodl genomic clones are isolated from a 129/SvJ genomic library (Stratagene) by screening with a rat Sodl cDNA probe.9 An approximately 7.2-kb Sac I genomic fragment, which contains the entire mouse Sodl gene, is isolated from clone 30 and used in the construction of the gene-targeting vector. To inactivate the mouse Sodl gene, the Smal and Hindlll restriction sites flanking the Smal-Hindlll fragment, which contains sequences from intron 1 to intron 4, are converted into Xhol sites by linker ligation, and then the fragment is inserted into the Xhol site of plasmid vector pPNT (Fig. 2). Similarly, linker ligation is also used to clone the EcoRl-Sall fragment containing the 3' flanking sequence of the gene into the BamHl site of the pPNT vector.

The Sodl targeting vector, in which exon 5 is deleted, is linearized by Hindlll digestion and transfected into R1 ES cells.9 Clones resistant to G418 and

9 P. Wang, H. Chen, H. Qin, S. Sankarapandi, M. W. Becher, P. C. Wong, and J. L. Zweier, Proc. Natl.

ganciclovir (Syntex) are screened by Southern blot analysis, using a probe 5' external to the genomic sequence present in the targeting vector (Fig. 2). Targeted clones are microinjected into C57BL/6 blastocysts according to standard procedure. Chimeric mice with near 100% chimerism are generated with Sodl knockout clone 5, and show 100% transmission of the 129/SvJ chromosomes.

Generation and Characterization of Sodl Knockout Mice

As shown in Fig. 2, exon 5 of the mouse Sodl gene (which encodes the C terminus of the protein, i.e., amino acid residues 120 to 154, which constitute both the structure and function of the active site channel and some of the flanking in-tron sequences) is replaced with the neomycin resistance cassette (neo). This also creates a new Pstl restriction site, resulting in a shorter Pstl genomic fragment from the targeted allele (12.5 kb) than from the wild-type allele (16.5 kb). Mice heterozygous (Sodl+l~) for the targeted allele are interbred to generate homozygous knockout (Sodl~'~) mice. Male and female Sodl~'~ mice grow normally and are apparently healthy under routine animal husbandry.

Inactivation of the functional mouse Sodl gene in mouse hearts by gene targeting is initially determined by RNA blot analysis. An approximate 50% reduction of Cu,ZnSOD mRNA is found in the hearts of Sodl+l~ mice compared with wild-type (Sodl+l+) mice. Furthermore, no Cu,ZnSOD mRNA can be detected in Sodl heart, indicating that the truncated Cu,ZnSOD or Cu,ZnSOD-neo fusion mRNA is degraded rapidly in the heart. Reduction of heart Cu,ZnSOD enzyme activity in Sodl+l~ and Sodl mice is also confirmed by SOD activity staining on a native polyacrylamide gel. Cu,ZnSOD activities in the hearts of Sodl+l+, Sodl+t , and Sodl~'~ mice are proportional to the mRNA levels in these mice.9 A decrease in Cu,ZnSOD activity apparently has no effect on the activity of other heart antioxidant enzymes such as MnSOD (Fig. 2, middle), catalase, glutathione peroxidase, and enzymes involved in the recycling of oxidized glutathione including glutathione reductase and glucose-6-phosphate dehydrogenase. Male and female Sodl+,+ and Sodl1' mice at 10 to 12 weeks of age are used for myocardial ischemia-reperfusion study.

Heme Oxygenase 1 Gene Knockout Mice

Heme oxygenase (HO) catalyzes the reaction for heme metabolism, yielding equimolar quantities of CO, iron, and biliverdin. The HO system consists of two isoforms: oxidative stress-inducible HO-1 (Hmox-1), also known as HSP 32, and the constitutive isozyme HO-2. Besides oxidants, a variety of other environmental stresses including heat stress, hypoxia, metals, endotoxin, and certain hormones can induce Hmox-1.10 Evidence suggests that Hmox-1 induction plays a role in

10 G. F. Vile and R. M. Tyrrell, J. Biol. Chem. 268, 14678 (1993).



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