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.
Objective 1, Stem technologies are being explored to provide an opportunity to cost-effectively create gene-specific modifications of food animal genomes via homologous genetic recombination and somatic cell nuclear transfer. In an effort to understand the cellular reprogramming process (making stem cells from differentiated cells), scientists at the University of Connecticut in collaboration with ARS scientists in Beltsville, Maryland used mouse fibroblasts to examine the role of epigenetic modifiers of chromatin (enzymes that add important biochemical groups to DNA in order to control gene expression) during stem cell development in culture. The findings indicate the role of specific regulatory genes involved in regulating epigenetic modification of DNA and the implications that these regulatory genes have on the reprogramming process. In keeping with interest in reprogramming and the use of embryonic stem cells, to understand gene expression during bovine embryo development, scientists at the University of Connecticut in collaboration with ARS scientists Beltsville, Maryland examined early bovine embryos for expression from both maternal and paternal genes. The expression levels of important developmental genes were compared to pig, mouse and human embryonic expression and significant differences were identified between the four species. In addition to important expression profiles for the bovine embryos, the data also provides a reference for expression profiles of important developmental genes in embryos produced using assisted reproductive biotechnologies. Despite a concerted effort from numerous labs worldwide, large mammal stem cell technologies have not yielded proven induced pluripotent stem cells that are useful in creating breedable “animals on the ground” as anticipated. Thus, the world-wide declining enthusiasm for livestock iPSC technologies (due to a lack of reports for breeding livestock produced from iPSCs for any species), has shifted the project focus to achieving genome editing utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Through direct injection into porcine embryos the lab has been able 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 and 30% genome edited bovine embryos at the blastocyst stage. The pig genomes were edited to remove genes that are important for sperm development and the resultant pigs are lacking all sperm. In Objective 2, the goal is to achieve transient transplantation of transgenic cells in newborn animals via simple injections in order to achieve temporary transgenic status that could mediate the expression of many beneficial proteins expressed in the growing animal that are not normally encoded by the animals genome. The transgenic cells would be killed via an inducible ‘kill gene’ before the animal go to slaughter. In an effort to identify candidate lines for use in this transient expression system, additional Swine Testis (ST; ATCC CRL-1746) epithelial cell lines were produced that were genetically engineered to express either human insulin-like growth factor-1 (hIGF1) or the antimicrobial protein human lipocalin-2 (hLCN2) or human growth hormone (hGH), each of which would confer enhancing growth characteristics or disease resistance to the animal. The new transgenic cell lines were tested by ELISA for the expression of the human factors; for hLCN2, one cell line was found to secrete robust amounts of hLCN2; for hIGF1 and the new hGH cell lines, several cell lines of each were shown to secrete robust amounts of hIGF1 or hGH into the culture medium. An animal care and use protocol was submitted and approved for the injection of ST cells transgenic for green fluorescent protein (GFP) expression into 21-day-old piglets. These tests will be conducted soon in order to see how long the ST cells will survive in piglets after injection into the piglet’s inguinal space. Several other transgenes vectors containing genes potentially beneficial to pig growth and health were prepared, or found, for the creation of more transgenic ST cell lines and included: the human antimicrobial protein pentraxin 3 (hPTX3), the human hepcidin antimicrobial peptide (hHAMP), the human myostatin inhibitor follistatin (hFS344), and the anti-staphylococcal protein lysostaphin which was mutated to function in eukaryotic cell expression. 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 underway to identify 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. 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. Toward this end, the genomes of 43 poultry strains of Clostridium perfringens were subjected to genomic DNA sequence analysis. Hundreds of potential anti-clostridial enzymes were identified and a family tree generated based on homology to known enzymes. Fifteen groups of enzymes were identified and 10 enzymes from distinct groups were tested for antimicrobial activity. Four enzymes (each from a distinct group) were purified from E. coli expression vectors and shown to be highly active against all 43 strains of Clostridium perfringens. In keeping with this goal, a lytic enzyme that targets Enterococcus faecalis was identified. The enterococcal VD13 bacteriophage infects E. faecalis. The phage genome was isolated and sequenced by a collaborating scientist at Ashland College, Ashland, Ohio. The VD13 phage endolysin gene was expressed and purified from Escherichia coli expression vectors and lytic specificity was demonstrated against 10 of 12 E. faecalis strains but no lytic activity against Enterococcus faecium, Staphylococcus aureus, E. coli, or streptococcal strains was detected. This enzyme is a potentially valuable alternative to antibiotics for agricultural applications.
1. Genome editing in single cell stage pig embryos yields an improved method to generate animal models. There is a requirement for large animal models such as pigs that can either serve as an alternative, or complement investigations from mouse models. Genome editing in single cell stage pig embryos yields an improved method to generate animal models. ARS scientists at Beltsville, Maryland in collaboration with University of Maryland scientists, have directed a change in the pig genome to a specific gene by direct injections of genome editing enzyme complexes into single cell stage porcine embryos resulting in gene targeted animals on the ground. The line of edited pigs carry a modified site that enables a commercially available recombinase enzyme to mediate the introduction of transgenes at this locus with an even greater frequency of success than without the modified site. The feasibility of inserting transgenes via the commercially available enzyme was also demonstrated. This new route for genome engineering in pigs through zygote injections should greatly enhance the creation of animal models and biotechnology applications in agriculture.
2. Identifying novel antimicrobials to target Clostridium perfringens, a poultry pathogen. Clostridium perfringens is a major necrotic enteritis causing bacterial pathogen in poultry, and a source of food poisoning and gas gangrene in humans, and can cause mild to severe enteritis in pigs. Due to the fear of farm to clinic transfer of antibiotic resistance genes, a ban on the use of antibiotic growth promotants in animal feed is pending resulting in a need for alternatives to antibiotics in animal feed. To address this need, an ARS scientist at Beltsville, Maryland has examined the genomes of 43 Clostridium perfringens isolates from chicken, and identified bacteriophage (viruses that infect bacteria) genomes embedded in the genomes of the bacteria. Hundreds of putative phage lytic enzyme genes were identified using molecular biological tools. Four of these enzymes were tested and shown to kill all 43 of the Clostridium perfringens isolates in lab assays but did not have a deleterious effect on other Gram positive or Gram negative species tested. This is an important step toward identifying novel replacements for novel antibiotic growth promotants that can be added to poultry feed.
Jiang, Z., Hong, D., Zheng, X., Donovan, D.M., Chen, J., Tian, X. 2015. mRNA levels of imprinted genes in bovine in vivo oocytes, embryos and cross species comparisons in humans, mice and pigs. Nature. 5:17898. doi: 10.1038/srep17898.
Talbot, N.C., Wang, L., Garrett, W.M., Caperna, T.J., Tang, Y. 2015. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines. In Vitro Cellular and Developmental Biology. 52(3):314-26.
Schmelcher, M., Pohl, C.S., Donovan, D.M. 2015. The streptococcal phage SA2 and B30 endolysins act synergistically and kill mastitis causing streptococci in milk. Applied and Environmental Microbiology. 99(20):8475-86.
Sandmaier, S.E., Nandal, A., Powell, A.M., Garrett, W.M., Blomberg, L., Donovan, D.M., Talbot, N.C., Telugu, B.V. 2015. Generation of induced Pluripotent Stem Cells from Domestic Goats - Capra hircus. Molecular Reproduction and Development. 82:709–721.
Kovalskaya, N.Y., Foster Frey, J.A., Donovan, D.M., Bauchan, G.R., Hammond, R. 2015. Expression of a bioactive bacteriophage endolysin in Nicotiana benthamiana plants. Journal of Microbiology and Biotechnology. 26:160-170.
Park, K., Park, C., Powell, A.M., Donovan, D.M., Telugu, B.P. 2016. Targeted gene knockin in porcine somatic cells using CRISPR/Cas ribonucleoproteins. International Journal of Molecular Sciences. 17(6).
Jiang, Z., Tang, Y., Zhao, X., Donovan, D.M., Tian, X. 2015. Knockdown Brm and Baf170, components of chromatin remodeling complex, facilitates reprogramming of somatic cells. Stem Cells and Development. 24(19):2328-36.
Shen, Y., Barros, M., Vennemann, T., Gallagher, D., Yin, Y., Linden, S.B., Heselpoth, R.D., Spencer, D.J., Donovan, D.M., Moult, J., Fischetti, V.I., Heinrich, F., Losche, M., Nelson, D.C. 2016. PlyC, a bacteriophage endolysin that is internalized by epithelial cells and retains bacteriolytic activity against intracellular streptococci. Stem Cells and Development. doi: 10.7554/eLife.13152.
Hoernig, K., Pithua, P., Williams, Iii, F., Donovan, D.M., Middleton, J. 2016. Evaluation of a lysostaphin-fusion protein as a dry-cow therapy for Staphylococcus aureus mastitis in dairy cattle. Journal Dairy Science Supplement. 99(6):4638-46.
Filatova, L.Y., Donovan, D.M., Ishnazarova, N., Foster Frey, J.A., Becker, S.C., Pugachev, V.G., Dmitrieva, N.F., Klyachko, N.L. 2016. A chimeric LysK-lysostaphin fusion enzyme lysing Staphylococcus aureus cells: A study of both kinetics of inactivation and specifics of interaction with anionic polymers. Enzyme and Microbial Technology. doi: 10.1007/s12010-016-2115-7.