Expression of hyg in Transgenic Mice Causes Resistance to Toxic Effects of Hygromycin B In Vivo

Aminoglycoside antibiotics are indispensable for treatment of serious bacterial infections, and despite careful attention to dosage regimens, nephrotoxicity and ototoxicity still cause concern. In the present study, we tested whether side effects of aminoglycoside therapy could be limited by expression of prokaryotic genes of antibiotic resistance in vivo. We characterized the acute and tissue-specific toxicity of hygromycin B in transgenic mice bearing the hygromycin B phosphotransferase (hyg) gene under control of a constitutive promoter. We characterized the tissue-specific expression of hyg mRNA and also investigated the acute toxicity of hygromycin B in hyg and wild-type mice. The hyg mRNA reached its highest levels in brain and reached intermediate levels in spleen, muscle, kidney, liver and testis. The lowest levels were detected in heart and lungs. The hyg expression in transgenic animals caused an 89-fold increase in the approximate lethal dose of hygromycin B compared with wild-type mice. Serum biochemical analysis of hyg and wild-type mice treated with lethal doses of hygromycin B indicated liver and kidney damage measured as ALT, AST and BUN. On the morphological level, these changes led to acute tubular nephrosis in wild-type mice and acute liver damage in hyg mice. Our results show that constitutive expression of the bacterial hyg gene in transgenic mice in vivo confers resistance to hygromycin B. Aminoglycoside antibiotics are indispensable for treatment of serious bacterial infections, and despite careful attention to dosage regimens, nephrotoxicity and ototoxicity still cause concerns (Lortholary et al., 1995). In the present study, we tested whether side effects of aminoglycoside therapy could be limited by expression of genes of antibiotic resistance in vivo. As a model system, we used aminoglycoside antibiotic hygromycin B and a transgenic mouse strain bearing the antibiotic resistance gene hyg under the control of the constitutive promoter Pgk1 (Johnson et al., 1995). Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus (Pettinger et al., 1953) that is active against both prokaryotic and eukaryotic cells. It has been shown that in eukaryotic cells, hygromycin B acts by interfering with protein synthesis, especially by inhibition of translocation, which is thought to be the result of its interaction with the eukaryotic ribosome displacing EF-2 from the ribosome or interfering with the activity of EF-2 and the stabilization of peptidyl-t-RNA bound to the ribosomal acceptor site (Gonzalez et al., 1978). Toxic effects of hygromycin B in tissue culture can be prevented by expression of hyg (Blochlinger and Diggelmann, 1984). This enzyme adds phosphate to position 7 of the destomic acid ring of hygromycin B, which results in complete loss of biological activity both in vitro and in vivo (Pardo et al., 1985). Therefore, hyg has been widely used as a positive selective marker in the construction of transgenic animals via ESC. The transgenic construct containing hyg is introduced into ESC. Then clones of ESC bearing recombinant DNA with hyg can be selected in medium containing hygromycin B. Although toxic effects of hygromycin B have been studied on the cellular level in tissue culture (see, for instance, Chen et al., 1995; Gonzalez et al., 1978; Pardo et al., 1985), we know of no previous reports on acute or tissue-specific hygromycin B toxicity in transgenic mice bearing hyg. For our experiments, we have used transgenic mice carrying hyg driven by the constitutive Pgk1 promoter (Johnson et al., 1995). These mice develop normally and do not exhibit any apparent abnormal pheno-