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Adenoviral-Mediated Transfer of a Lysostaphin Gene into the Goat Mammary Gland

Department of Animal Science College of Agricultural & Life Sciences University of Vermont, Burlington 05405

Corresponding author:
D. E. Kerr; e-mail:
dkerr{at}zoo.uvm.edu.

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln^{125, 232}-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding ß-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of ß-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln^{125, 232}-lysostaphin (0.8 µg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10¹⁰ plaque-forming units (pfu) of Ad-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of ß-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10¹⁰ pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of ß-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln^{125, 232}-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 µg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln^{125, 232}-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.

Abbreviation key: Ad-lacZ = recombinant adenoviral vector containing the Escherichia coli lacZ gene, Ad-lys = recombinant adenoviral vector containing a modified lysostaphin gene, BME-UV = bovine mammary epithelial cell line—University of Vermont, hGH = human growth hormone, lacZ = E. coli gene encoding ß-galactosidase, X-gal = 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.

Key Words: mastitis • adenovirus • lysostaphin • gene therapy

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				J. X. Zhang, S. F. Zhang, T. D. Wang, X. J. Guo, and R. L. Hu Mammary Gland Expression of Antibacterial Peptide Genes to Inhibit Bacterial Pathogens Causing Mastitis J Dairy Sci, November 1, 2007; 90(11): 5218 - 5225. [Abstract] [Full Text] [PDF]

				W. Fan, K. Plaut, A. J. Bramley, J. W. Barlow, S. A. Mischler, and D. E. Kerr Persistency of Adenoviral-Mediated Lysostaphin Expression in Goat Mammary Glands J Dairy Sci, March 1, 2004; 87(3): 602 - 608. [Abstract] [Full Text] [PDF]