OBJECTIVE 1: Develop techniques for genetic modification and genetic engineering of food animals including pluripotent stem cells, somatic cell nuclear transfer and genome editing technologies. 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 either be expressed in food animals with in vivo activity via transgenic technology, or delivered via feed additives consisting of either the purified agent or extracts/preparations of biofermenation organisms expressing the recombinant antimicrobial (e.g. Lactococcus lactis, yeast). 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. 3C. Identify, isolate, and characterize multiple bacteriophage genes that express proteins with unique lytic activities against Clostridium perfringens and express these constructs in Saccharomyces cerevisiae. Verify that these lytic activities are maintained in the yeast-expressed proteins and test for effects on enteric C. perfringens and other gut flora when the transgenic yeast are fed to chickens.
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 project objective will 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 project objective will also 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, the world-wide declining enthusiasm for livestock iPSC technologies (due to a lack of reports for breeding livestock produced from iPSCs for any species), the focus has switched to achieving genome editing utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system through direct injection into early bovine and porcine embryos to introduce DNA modifications at a single, predicted site in the genome. In collaboration with a University of Maryland scientist in shared facilities with ARS scientists in Beltsville, Maryland, the bovine prion gene and an important reproduction gene in pigs were targeted in single cell embryos. Scientists found nearly 50% of the seven-day bovine embryos harbor biallelic genomic modifications of the prion gene; thus the modifications should result in the knockout of prion gene function. Knockout pigs have been produced for the reproduction gene and are being further studied by collaborators at University of Washington. For Objective 2, an additional Swine Testis (ST; ATCC CRL-1746) epithelial cell line was produced by ARS scientists that was genetically engineered to express human growth hormone and green fluorescent protein. The new transgenic cell line was found to express human growth hormone mRNA and the cells of the cell line fluoresced green when viewed with blue light. The additional transgenic ST cells will be injected into day-old piglets to see how long they can survive in the body and to see if they can produce enough human growth hormone to be detectable in the piglet’s blood. For Objective 3, the optimal mammalianized triple-acting staphylolytic protein fusion (with a protein transduction domain to transport the protein into mammalian cells) was verified to have three active domains via mass spectrophotometry and deletion analysis. Intracellular activity of this mammalianized construct against Staphylococcus aureus is ongoing. The genomic annotation of the staphylococcal strains that developed resistance to our triple-acting lytic proteins has been completed and polymorphisms compiled. Comparison of the polymorphisms between strains is underway. Advances have been made to create antimicrobial surfaces through the use of polydopamine-assisted immobilization of bacteriolytic enzymes to innate surfaces such as glass and plastic. These surfaces should prevent biofilm formation and could be useful tools for food safety applications (food wrap), milking machines, or other veterinary and human health care applications (catheters). In addition, a novel anti-Clostridium perfringens antimicrobial enzyme was produced by ARS scientists that has high pathogen-specificity as well as improved thermostability is to be tested for its utility as an animal feed additive.
1. Antimicrobial surfaces created by attaching enzymes that kill bacteria. There is an urgent need for antimicrobial surface treatments that are semi-permanent, can eradicate both biofilms (growing masses of bacteria that attach to surfaces) and planktonic (growing in liquid) bacterial pathogens over long periods of time, and that do not allow resistant strain development. ARS scientists at Beltsville, Maryland, have recently demonstrated a simple, robust method to attach antibacterial enzymes to a variety of surfaces to generate surfaces that kill bacteria. The immobilization of the enzymes was carried out under conditions compatible with mammalian cells that maintains antimicrobial activity and kills both human and animal strains of Staphylococcus aureus in a few minutes. The simultaneous use of three distinct lytic activities in one enzyme can reduce the development of antimicrobial-resistant strains. Use of this type of antimicrobial coated surface has utility to prevent bacterial biofilm formation by the targeted pathogens. This strategy may be utilized to develop antimicrobials against numerous pathogens in addition to Staphylococcus aureus, and is expected to be useful for both food safety (food packaging) as well as animal (e.g., milking machines) and human health concerns (catheters).
2. Anti-Clostridium perfringens antimicrobial with high thermostability created by enzyme fusion. Clostridium (C.) perfringens is a notorious poultry pathogen that causes necrotic enteritis (a poultry gut disease) that results in billions of dollars in loss to the poultry industry annually, and is currently controlled by antibiotics in animal feed. Antibiotics in animal feed are being phased out world-wide due to concerns for antibiotic resistance transfer from farm to clinic. It is important for feed additives to be heat-resistant due to the high temperatures used to process animal feed. ARS scientists in Beltsville, Maryland, and Athens, Georgia, engineered an antimicrobial enzyme (endolysin) from a bacterial virus (bacteriophage) that is specific for C. perfringens and is more heat-tolerant than the parental C. perfringens-specific enzyme from which it was derived. An antimicrobial enzyme from a bacteriophage that infects Geobacillus, a heat-loving bacterium (a thermophilic bacterium), was fused to a C. perfringens bacteriophage enzyme melding both the specificity of the C. perfringens phage endolysin and the thermostability of the Geobacillus endolysin into one enzyme. This is an important step toward identifying novel replacements for antibiotic growth promotants that can be added to poultry feed.
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