Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland.
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, Gln125,232-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 beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/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 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-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 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-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.
Department of Animal Science, College of Agricultural & Life Sciences University of Vermont, Burlington 05405, USA.
W
W
Fan
K
K
Plaut
A J
AJ
Bramley
J W
JW
Barlow
D E
DE
Kerr
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D015511:Goat Diseases / Q000382:microbiology / Q000517:prevention & control*; D006041:Goats; D008248:Lysostaphin / Q000096:biosynthesis*; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000502:physiology; D008413:Mastitis / Q000517:prevention & control / Q000662:veterinary*; D010455:Peptides; D013203:Staphylococcal Infections / Q000517:prevention & control / Q000662:veterinary; D013211:Staphylococcus aureus / Q000235:genetics*
D000900:Anti-Bacterial Agents; D010455:Peptides; D008248:Lysostaphin