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, putative goat iPSC (giPSC) cell lines were characterized for their pluripotent differentiation potential. Immunocytochemical analysis showed that the giPSC cell lines could produce cells that were positive for the neuronal protein markers vimentin, alpha-internexin, and neurofilament protein. Genome editing of livestock allows engineered DNA modifications at a single, predicted site in the mammalian genome. The bovine prion gene has been targeted in single cell bovine embryos. DNA from embryos that survived seven days harbor biallelic genomic modifications (in both gene copies) of the prion gene; the modifications should result in the knockout of prion gene function. The technology will be further demonstrated by transferring these genome edited embryos to recipient cows with the goal to produce prion gene knockout animals. For Objective 2, two porcine immortal cell lines, TD-1 pig fibroblasts and Swine Testis (ST) epithelial cells (ATCC CRL-1746) were tested for efficiency in producing stable transfectants using mammalian vectors expressing the neomycin-resistance gene and human growth hormone (GH) tagged with green fluorescent protein. The expression of human GH from the TD-1 and ST transfectants is being assessed. Subclones of TD-1 and ST cells containing the empty expression vector, and subclones of NIH/3T3 mouse fibroblasts, transfected with the same human GH expression vector, were also created to act as negative control and xenogeneic transplantation control cell lines, respectively. PICM-19 cells readily ingested sufficient iron nanoparticles to allow magnetic resonance imaging tracking of the nanoparticle-laden-cells. They did not suffer functional deficits or morphological changes. The iron nanoparticle-loaded PICM-19 cells will be followed in vivo by MRI after being injected into the peritoneum of piglets to assess the location and survival of the transplanted cells over time. The work will be conducted in collaboration with a researcher at Michigan State University, East Lansing, Michigan. For Objective 3, bacteriophage endolysins known to be active in milk were used to create fusion constructs for testing of both the maintenance of three lytic activities and for antimicrobial activity in milk. Most show weak activity in milk. The milk-active candidates are being tested for activity against staphylococcal mastitis pathogens other than Staphylococcus aureus. In order to better understand putative resistance mechanisms to phage lytic proteins, Staphylococcus aureus isolates that were resistant to triple-acting antimicrobial enzymes were subjected to DNA sequence analysis under a competitive NIAID funded project. The annotation of these genomes is being completed in collaboration with a University of Maryland scientist. In preparation for creating a transgenic mouse expressing a triple-fusion construct, the construct was analyzed bioinformatically for glycosylation sites and the sites were mutated to disallow glycosylation within eukaryotic cells. The mutant construct was demonstrated to maintain its three lytic activities and is being optimized for expression in cultured bovine cells.
1. Lactobacillus bacteriophage endolysins expressed in yeast protect ethanolic fermentations. Lactobacillus bacteria can contaminate biofuel (ethanol producing) fermentations and cause premature death of the fermentative yeast that results in suboptimal ethanol production. ARS scientists in Beltsville, Maryland and Peoria, Illinois have expressed Lactobacillus bacteriophage endolysins (enzymes that kill Lactobacillus) in fermentative yeast and have demonstrated the efficacy of these yeast to kill the Lactobacillus contaminants, and maintain conditions required for optimal ethanol production. Endolysin expression did not appear to be a significant burden on the yeast as evidenced by yeast growth and ethanol production during the mock fermentations. This approach could save the biofuel industry the cost and environmental burden of treating contaminated fermentations with antibiotics and the expense of cleaning the fermentation facilities after a contaminated fermentation.
2. Staphylococcal phage lytic enzymes for treating intraocular eye infections. The treatment of endophthalmitis, an eye infection of the vitreous humor (the largest eye cavity; between the lens and the retina), is becoming very challenging due to the emergence of multidrug-resistant bacteria, including Staphylococcus aureus. The development of novel therapeutic alternatives for endophthalmitis treatment is essential. In collaboration with scientists at Wayne State University, ARS scientists from Beltsville, Maryland have evaluated the therapeutic potential of an enzyme that kills Staphylococcus aureus in a mouse model of Staphylococcus aureus endophthalmitis. The enzyme exhibited strong killing activity against Staphylococcus aureus in vitro, as evidenced by a complete inhibition of bacterial growth and disruption of biofilms. The injection of the enzyme into the mouse eye significantly improved the outcome of staphylococcal endophthalmitis, preserved retinal structural integrity, and maintained visual function. The treatment significantly reduced the bacterial burden and the levels of the mouse immune system response in the treated eye. These results indicate that the administration of this type of enzyme can attenuate the development of bacterial endophthalmitis in mice, and could save both human and veterinary health care costs associated with failed treatment due to drug resistant strains of pathogen.
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