OBJECTIVE 1: Develop stem cell techniques for genetic modification and genetic engineering of food animals including pluripotent stem cells, somatic cell nuclear transfer and gene product genetic engineering. 1A. Establish normal, functionally immortal, ungulate iPSC lines that lack genomic integration of either viral vectors or RF genes in the ungulate genome. 1B. Demonstrate the use of bovine iPSC lines for genetic engineering of cattle via chimerism, in vitro transgenesis, and NT. OBJECTIVE 2: Improve porcine production and product traits through expression of specific gene products via transient allogeneic cell transplantation of genetically engineered porcine cells into newborn pigs. 2A. Determine culture conditions for an immortal porcine cell line that enables the cells to be transplanted into pigs without hyperacute rejection. 2B. Determine the capacity of the cells of an immortal porcine cell line to survive in vivo after allogeneic cell transplantation into neonatal pigs. 2C. Determine if hGH-expressing transplanted cells affect the growth of neonatal pigs. OBJECTIVE 3: Reduce the emergence of drug resistance in common pathogens of food animals by developing recombinant antimicrobial gene constructs that can be expressed in food animals with in vivo activity via transgenic technology. 3A. Create, identify and test mutant versions of existing triple-acting recombinant antimicrobials for high lytic activity in a milk environment. 3B. Develop eukaryotic expression cassettes for triple-acting bactericidals and test for antimicrobial efficacy in cultured mammary cells and mammary glands of transgenic mice.
The project will develop approaches for expressing new gene products in livestock that can be utilized to improve food animal production/efficiency, enhance traits, maintain strain- or breed-specific genetics, minimize disease susceptibility or improve product safety. The first objective will be the production of porcine and bovine induced pluripotent stem cell (iPSC) cell lines, or other long-lived cell lines, that survive sufficiently long in culture to enable targeted gene replacements to be effected in cattle and pigs. The cell lines may also serve as an immortalized version of cryobanked material for the preservation of breeds. As an alternative to permanently modifying the livestock genome, the second project objective will be to affect the phenotype of pigs via allogeneic transplantation of cultured pig cells that are transgenically modified to secrete specific proteins that confer a benefit to animal production traits, e.g., growth status, and harbor an inducible ‘suicide’ gene for ablation of the cells prior to animal harvest. The third objective will be to develop triple-acting antimicrobial gene constructs as model transgenes to prevent bovine mastitis caused by Staphylococcus aureus. An expansion of prior lysostaphin work, the constructs will be designed with three unique staphylolytic activities as a strategy to reduce the development of resistant bacterial strains. Additionally, a protein transduction domain will be incorporated into the constructs to allow the antimicrobial protein to penetrate mammary cells and eradicate intracellular S. aureus that are typically associated with chronic mastitis infections.
For Objective 1, we have developed specialized stem cell lines from food animals that can be used to define both functional roles of economically important genes and develop better techniques for enhancing livestock genetics via the transfer of genes within and across species. Embryonic stem cells (ESC) have a key advantage over other cells in that they can be maintained for very long periods of time in culture; this enables their use for the most complex genetic manipulations. The technology, once developed, will aid traditional animal production research applications and be used to decrease the environmental footprint of animal production, improve animal health, well-being and resistance to disease, and enhance food safety. We tested six factors and specialized small RNAs (microRNAs) that resulted in the production and maintenance of cow, pig and goat ESC-like stem cells in culture. However, these stem cells exhibited limited pluripotency, i.e., the ability to differentiate into many different cells types of the body. Authentic ESC are able to turn into virtually any of the different cells types of the body when experimentally injected into a mouse, for formation of a tumor called a teratoma. To date, the teratomas produced from the cow, pig, and goat ESC-like stem cells gave rise to only a few cell types indicating that the stem cells are not truly pluripotent. In Objective 2, serum-free culture conditions for the in vitro growth of pig cells were devised, tested and found to be successful. Also, the in vitro growth of pig cells using pig-serum-containing culture medium, in place of the conventional fetal-bovine-serum-containing culture medium, was tested and found to support growth of the cells. These results indicate that pig cells can be grown without foreign proteins (i.e., proteins from other species) so that transplantation of in vitro-cultured pig cells into live pigs will likely survive and not be rejected immediately by the recipient pig’s immune system. In a parallel and more direct effort to create agents to improve animal health and reduce disease resistance, we have made significant progress toward Objective 3 by identifying bacterial virus proteins that can be engineered to express three simultaneous antimicrobial actions and thereby preventing the bacteria from escaping the killing action. We have modified our antimicrobials such that they are transported across the mammalian cell wall and have demonstated killing of bacteria that reside inside cultured cells and staphylococcal bacteria that reside in biofilms (biofilms are bacterial cultures that can form thin layers of cells on catheters or other inert or biological surfaces). We have shown that these engineered proteins can cure three different infection models in mice. Additional success includes demonstrating that we can modify critical amino acid residues in the antimicrobial proteins to allow a higher level of production and release of the proteins in mice while maintaining the antimicrobial activities.
1. Identification of Engineered Antimicrobial Proteins that Eradicate Staphylococcal Mastitis Pathogens in Live Animals. The United States Dairy industry loss due to mastitis (infections of mammary glands) exceeds $2 billion annually. Mastitis is also responsible for the greatest use of antibiotics on the dairy farm despite a world-wide effort to reduce agricultural antibiotic use as a means to reduce the world-wide increase of resistance to existing antibiotics. The bacterial pathogen, Staphylococcus aureus (S. aureus), can evade most conventional antibiotics by invading and residing inside the cells (intracellularly) of the cow mammary gland, leading to chronic infection and increased elimination of S. aureus infected animals. Conventional antibiotics do not kill intracellular pathogens. Thus, novel antimicrobials that were also effective at killing intracellular bacteria would benefit the dairy industry world-wide for the treatment of mastitis and help to replace the failing antibiotics that are facing high levels of resistant strain development world-wide. ARS scientists in Beltsville, Maryland demonstrated that an engineered antimicrobial protein which harbors a domain to facilitate transport of the antimicrobial protein across the mammary cell walls to intracellular spaces (protein transduction domain), reduces S. aureus that reside within cultured mammary cells. They fused the protein transduction domain to a previously engineered antimicrobial protein with three distinct enzyme activities. The top candidate engineered antimicrobial protein, for the eradication of intracellular S. aureus in cultured cell assays, also showed the ability to reduce the S. aureus infection 1000-fold in a mouse mastitis model. This technology presents an alternative mastitis treatment that should cure bovine mastitis and reduce the need for conventional antibiotic use on the dairy farm.
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Schmelcher, M., Korobova, O., Schischkova, N., Kiseleva, N., Kopylov, P., Pryamchuk, S., Donovan, D.M., Abaev, I. 2012. Staphylococcus haemolyticus prophage endolysin relies on CHAP endopeptidase lytic activity for lysis from without. FEMS Microbiology Letters. 162(2-3):289-298.
Rodriguez-Rubio, L., Martinez, B., Donovan, D.M., Garcia, P., Rodriguez, A. 2013. Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation. Applied Microbiology and Biotechnology. 8(1):e54828.
Mao, J., Donovan, D.M. 2013. Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme. Applied and Environmental Microbiology. 342(1):30-36.
Rodriguez-Rubio, L., Martinez, B., Rodriguez, A., Donovan, D.M., Götz, F., García, P. 2013. Undetectable bacterial resistance to phage lytic proteins from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. PLoS One. 8(5):e64671.
Talbot, N.C., Caperna, T.J., Garrett, W.M. 2013. Development, characterization and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19. Journal of Animal Science. 91:66-77.
Rogee, S., Bouquet, J., Barnaud, E., Pavio, N., Talbot, N.C., Caperna, T.J. 2013. New models of hepatitis E virus replication in human and porcine hepatocyte cell lines. Journal of General Virology. 94(3):549-558.
Roach, D.R., Khatibi, P.A., Bischoff, K.M., Hughes, S.R., Donovan, D.M. 2013. Bacteriophage-encoded lytic enzymes control growth of contaminating Lactobacillus found in fuel ethanol fermentations. Biotechnology for Biofuels. 6(1):20.