Project Number: 8042-31000-103-00-D
Project Type: In-House Appropriated
Start Date: Jul 24, 2012
End Date: Jul 23, 2017
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.