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.
The project is terminating in July 2017. There has been significant progress in each of the three objectives. The goal to create genome edited livestock has been realized with numerous models of genome edited pigs. The goal to create transiently transgenic pigs is similarly nearly completed with ‘live cell’ injections into neonatal pigs ongoing. The goal to identify novel antimicrobials to treat chickens with necrotic enteritis has been successful and gained industry interest with funding from an industry partner via a CRADA. In this CRADA, the novel antimicrobials will be tested in chickens in 2017. 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 (GE) utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system through direct injection into 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 ABBL, we have achieved 100% biallelic genome edited pigs on the ground. As described below in the accomplishments, these efforts have created multiple lines of GE knockout and/or transgenic (Knock In) pigs. Most impressive is the ability to first create cloned pig embryos (from fibroblast cells and abattoir-derived oocytes) perform GE on those cloning-derived embryos just hours after completing the somatic cell nuclear transfer, and to then transfer those embryos into recipient sows and generate GE pigs from newly cloned and edited embryos. This allows functional genomics to be performed on rare breeds of pigs, for which it is not feasible to maintain a herd of sows to provide embryos. A type of cell which occurred at high frequency in our bovine reprogramming experiments from 3-4 years ago was characterized. These cells, resulting from the reprogramming of bovine fibroblasts, were identified to be trophectoderm cells, the first differentiated cells of the early pre-implantation bovine embryo. A manuscript describing this result was published this year. Pig fibroblast reprogramming was also undertaken 3-4 years ago, and the resulting 24 cell lines of cells with characteristics of induced pluripotent stem cells (iPSC) have been examined for their embryonic stem cell (ESC) characteristics. A manuscript describing the results of the study is in preparation. For Objective 2, the ST pig cells genetically engineered to carry and express the green fluorescent protein (GFP, a marker protein that glows green in the cells under blue light) were injected subcutaneously into neonatal piglets to see if they would survive for up to 3 weeks under the skin of the piglets. The ST-GFP cells were found to survive at the injection site for 10 days post-injection (transplantation), but long time point assessments were complicated by the technical difficulty of identifying the injection site after 3 weeks of piglet growth. Further test of cell transplant survival will be pursued using injection site marker dye and co-injection of fluorescent microbeads with the ST-GFP cells. For Objective 3, 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 transcriptome of these strains is still a work in progress, with collaborators who are very interested but have limited resources. One comparison is completed and candidate genes identified with altered gene expression that might be responsible for the resistance phenotype. In preparation for making a transgenic animal expressing a triple-acting anti-staphylococcal protein (with three lytic activities in one protein), a construct was designed for expression in eukaryotic cells (codon optimized, and all of the glycosylation sites were modified), and shown to be active at eradicating S. aureus when expressed and secreted from cultured cells. However the expression from the cultured cells is very low and we are trying to improve the levels achieved prior to attempting animal expression. In order to reduce the emergence of drug resistance in pathogens that negatively impact food animal production, one goal of this project is to develop recombinant antimicrobial constructs that can be delivered via feed additives. Feed additives need to be tolerant of the high temperature rigors of feed processing. Toward this end, the genes for four thermophilic phage endolysins from public datasets were fused to the cell wall binding domains of Clostridium perfringens phage endolysins, purified and tested for activity against C. perfringens. Three of the four fusion constructs are lytic for multiple strains of C. perfringens. These enzymes are potentially valuable alternatives to antibiotics for agricultural applications to treat C. perfringens that can cause intestinal disease in piglets, chickens and bovine calves.
1. Identification and characterization of bovine placental cells resulting from the cultured cells. There is a need to better understand early embryonic gene expression in livestock, as it pertains to successful reproduction and early embryonic development in the uterus. ARS scientists in Beltsville, Maryland, were able to redirect the development of bovine connective tissue cells (fibroblasts) by expression in the fibroblasts of several “reprogramming genes”. Showing that the reprogramming of bovine cells may be different when compared to the reprogramming results in other species of animals, such as the pig, mouse or human.
2. It is believed that genetically modified livestock will be important in feeding the world in 2050 when the human population of Earth is predicted to exceed 9 billion. The domestic pig is an agriculturally important food species and a proven model of human physiology. ARS scientists from Beltsville, Maryland, in collaboration with scientists from University of Maryland, College Park, Maryland, Renovate Biosciences Inc., Reisterstown, Maryland, Washington State University, Pullman, Washington, The Roslin Institute, Edinburgh, Scotland, and Genus PIC, DeForest, Wisconsin, used genome editing technology to rapidly modify the pig genome and eliminate the function of an economically important gene (NANOS2 knockout) known to be essential for sperm maturation. The ability to modify the genome and to rapidly generate genetically modified animals is desirable for both basic science, and for demonstrating the role of important genes in livestock production.
3. Gene editing to derive animal model to study obesity. The availability of tools (CRISPR/CAS9) to edit the genome of an animal by making site-specific alterations with relative ease in the single cell embryo allow the generation of animal models to study specific diseases or health issues of concern to both agriculture and biomedicine. The Ossabaw pig breed has often been used as a model of human obesity because it over eats and its regulation of insulin is suppressed; however, it is difficult to get this breed from research suppliers. ARS scientists, Beltsville, Maryland, in collaboration with scientists from University of Maryland, College Park, Maryland used genome editing tools in combination with cloning technology, like that used in the cloning of Dolly the sheep, to repress insulin signaling in a standard pig breed and derive pigs with Ossabaw-like traits, that is over eating and a repressed insulin production. This pig model will be used to study the role of insulin signaling in an animal that is prone to obesity. Animals for a specific disease model can now be produced at will without the need to manage.
Park, K., Powell, A.M., Sandmaier, S.E., Kim, C., Mileham, A., Donovan, D.M., Telugu, B.P. 2017. Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes. Stem Cells and Development. doi: 10.1038/srep42458.
Ki-Eun, P., Kaucher, A., Powell, A.M., Wagas, M., Sandmaier, S., Oatley, M.J., Park, C., Tibary, A., Donovan, D.M., Blomberg, L., Lillico, S., Whitelaw, B., Mileham, A., Telugu, B., Oatley, J.M. 2017. Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Nature Communications. doi: 10.1038/srep40176.
Sheets, T.P., Park, C., Park, K., Powell, A.M., Donovan, D.M., Telugu, B.P. 2016. Somatic cell nuclear transfer followed by CRIPSR/CAS9 microinjection results in highly efficient genome editing in cloned pigs. International Journal of Molecular Sciences. doi: 10.3390/ijms17122031.
Swift, S., Rowley, D.T., Young, C., Franks, A., Hyman, P., Donovan, D.M. 2016. Characterization of the endolysin from the Enterococcus faecalis bacteriophage VD13. FEMS Microbiology Letters. doi: 10.1093/femsle/fnw216.
Verbree, C.T., Datwyler, S.M., Eichenseher, F., Meile, S., Donovan, D.M., Loessner, M.J., Schmelcher, M. 2017. Identification of peptidoglycan hydrolase constructs with synergistic staphylolytic activity in cow's milk. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.03445-16.
Donovan, D.M., Ealy, A.D., Powell, A.M., Caperna, T.J., Blomberg, L., Garrett, W.M., Sparks, W.O., Talbot, N.C. 2017. Bovine trophectoderm cell lines induced from bovine fibroblasts with reprogramming factors. Reproduction. 84(6):468-485.