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Fig. 1. Southern analysis of human MnSOD gene in transgenic mice. Five micrograms of genomic DNA from the tail of each transgenic mouse was digested with restriction enzyme Pstl. After digestion, the DNA was separated on a 0.8% (w/v) agarose gel, transferred to nitrocellulose, and probed with 32P-labeled human MnSOD cDNA.

Alternatively, the presence of the human MnSOD transgene in the mouse genome can be detected by polymerase chain reaction (PCR). In this case the same genomic DNA used for Southern analysis can be used in the PCR. To assure that a negative result is not due to failure in the amplification reactions, the mouse MnSOD gene is also amplified from the same DNA sample. PCR is carried out in a 50-/il reaction mixture containing 20 mM Tris-HCl (pH 8.8), 2 mM MgS04, 10 mM KC1, 10 mM (NH^SO* 0.1% (v/v) Triton X-100, nuclease-free bovine serum albumin (BSA, 1 mg/ml), 10 mM each of dATP, dCTP, dGTP, and dTTP, 0.2-0.5 ¡i 1 of Taq DNA polymerase, and 5 ¡i\ of genomic DNA. The oligonucleotide sequences of the primer sets are as follows:

Human MnSOD (positions 93-280): 5'-AGC ATG TTG AGC CGG GCA GT-3' 5'-AGG TTG TTC ACG TAG GCC GC-3' Mouse MnSOD (positions 8-280):

5'-AAC GGC CGT GTT CTG AGG AG-3' 5'-AGG TGG TTC ACG TAG GCC GC-3'

Each PCR product of MnSOD is then subjected to thermal cycling. Human MnSOD PCR product is initially denatured at 94° for 2 min, followed by 29 cycles (30 sec of denaturation at 94°, 15 sec of annealing at 65°, 20 sec of extension at 72°), and finished with a final extension at 72° for 7 min. Mouse MnSOD PCR product is initially denatured at 94° for 2 min, followed by 34 cycles (3 sec of denaturation at 94°, 30 sec of annealing at 60°, 40 sec of extension at 72°), and finished with a final extension at 12° for 7 min. The PCR products are then analyzed on a 1% (w/v) agarose gel in Tris-acetate buffer with ethidium bromide staining. The products of human MnSOD PCR and mouse MnSOD PCR are 187 and 272 bp, respectively.

Southern blot analysis screening of MnSOD transgenic mice is preferred over PCR analysis because the presence of bands of the predicted size(s) on a Southern blot clearly indicates a positive transgenic mouse, and under appropriate conditions less than one copy per cell of gene can be detected, whereas PCR can easily give a false-negative result. Also, contamination of a mouse DNA sample with plasmid DNA is likely to produce bands that differ in size from the expected bands on a Southern blot, while contaminating plasmid could cause a false positive on PCR. In addition, a Southern blot provides immediate information about the structure and integrity of the inserted DNA sequences. However, Southern blot analysis is subject to errors due to such factors as nonuniform transfer of DNA to the filter and incomplete digestion with the restriction enzyme. The choice of restriction enzyme and hybridization probe for the analysis of the integrated DNA is important.

Northern Analysis of MnSOD mRNA

The expression of steady state mRNA from the human MnSOD gene in various issues of transgenic mice can be demonstrated by Northern analysis. The levels of human MnSOD mRNA in tissue are detected by Northern analysis after isolation of total RNA by the guanidine isothiocyanate method.9 Northern analysis of MnSOD mRNA is straightforward and hybridization conditions used in Southern analysis can be applied here. The most critical factor in the success of mRNA analysis is the quality of RNA used. Although many commercial kits for RNA isolation can be used, guanidine isothiocyanate with cesium chloride centrifugation as described by Chirgwin et al.23 works best for tissues with a high level of ribonuclease. Figure 2 shows Northern analysis of MnSOD mRNA isolated from the skin of transgenic and control mice.

Western Blot and Activity Gel for MnSOD

Western blot allows for direct detection of MnSOD via its recognition by specific antibody raised against the purified protein. Standard conditions for electrophoresis and transfer of proteins are used in our study. However, it is important to note that the specificity of the antibody is important for this assay. The antibody raised against human MnSOD used in our work was kindly provided by L. Oberley at the University of Iowa (Iowa City, IA). Although the antibody also recognizes mouse MnSOD, it consistently provides a single shape band of human MnSOD protein on a blot. To visualize the activity of the MnSOD protein on the

23 j. M. Chirgwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, Biochemistry 18,939 (1979).

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