2009 Annual Report 1a.Objectives (from AD-416) 1. Improve the predictability of transgene expression through addition of genetic control elements and utilization of endogenous gene regulation. Devise strategies for stoichiometric coordinated expression of multi-cistronic transgenes. 1.A. Develop a 'state of the art' transgene expression cassette for controlled mammary expression that is efficacious when delivered either by pronuclear injection or transfection. 1.B Develop lentiviral vector strategies for improved transgene integration and embryo survival. 1.C. Optimize a mammary gland specific expression vector for the coordinated expression of multiple transgenes. 2. Develop a strategy for reducing drug resistance in S. aureus by identifying antimicrobial peptides that exhibit alternative modes of action. 2.A. Identify S. aureus surface structures that participate in resistance to peptidoglycan hydrolases and test triple acting fusion peptidoglycan hydrolases for their ability to evade the mechanisms identified. 2.B. Test candidate peptides in transgenic mice for their ability to protect against S. aureus infection. 3. Assess the merits of cloning and genetic enhancements in dairy cattle by evaluating clones and transgenic cattle and their offspring in a production setting with appropriate contemporaneous comparator animals. 3.A. Determine if a mammary gland specific transgene product can be detected in tissues other than the mammary gland throughout development of the transgenic cattle. 3.B. Determine if the cloning process substantially alters the epigenetic marking patterns between nuclear donor cells and their derived clones and the offspring of the clones. 3.C. Determine if clones, which are produced asexually, exhibit normal meiosis during the formation of their gametes. 1b.Approach (from AD-416) Objective 1: Create three 'LG-luciferase transgene constructs that are designed to utilize different strategies to regulate transgene expression. Analyze these constructs for reproducible, inducible, lactation-specific expression in cultured MAC-T cells. Verify the MAC-T result in mammary glands by creating transgenic mice with the construct demonstrating the most reproducible expression. Because of the large size of the BAC construct, the luciferase reporter will have to be introduced by homologous recombination rather than by conventional cloning techniques. The same homologous recombination knockin vector will be used to build the endogenously controlled promoter trap transgene. Objective 2: PCR cloning will be utilized to fuse the mature form of lysostaphin to the phi11 endolysin and to the LysK endolysin. These fusion constructs will have CHAP endopeptidase, amidase and glycyl-glycyl endopeptidase domains. Imidazole step gradient on a nickel column will yield a phi11 endolysin-lysostaphin that is free of contaminating protein, to be used against S. aureus in a turbidity assay. A similar imidazole step gradient will be used to purify the LysK endolysin-lysostaphin construct. To determine if the endolysins (CHAP or amidase) contribute to cell lysis, each domain will be subjected to site-directed mutagenesis to inactivate domains one at a time. Objective 3. Tissues will be collected at slaughter from lactating transgenic females and pregnant females from each of the three lines, and from non-transgenic lactating females (negative control). Tissues will also be collected from at least one sexually mature bull from each of the lines. The tissues will be stored at -80C or 0.5 gm will be added to 2.5 ml of RNAlater, minced and then frozen until processed. The tissues to be collected include brain, blood, gonads, heart, kidney, muscle, liver, lung, lymph node, salivary gland, and spleen. All samples will be assayed for lysostaphin transgene mRNA expression (normalized to beta-actin expression), protein and biological activity as we have previously described. For gene-specific DNA methylation and histone acetylation, the bisulfite sequencing and Chromatin precipitation assay (CHIP) will be used. The methylation of cytosines in CpG dinucleotides will be determined from the sequence information of at least 10 independent clones. For the CHIP assay, antibodies specific to acetylated histones will be incubated with DNA in cells after fixation. Amplification of specific genes will be conducted by real-time PCR after bound DNA was precipitated by centrifugation. The amount of histone acetylation can be determined by the levels of amplification. Synaptonemal complex structure and chiasmata localization will be characterized by immunofluorescent staining of SCP3 (synaptonemal complex lateral element protein. 3)and MLH1 (MutL homolog 1 mismatch repair protein) foci. 3.Progress Report To improve reliability of transgene expression we plan to target specific genome loci. Gene targeting must be performed in cells capable of self-renewal and able to generate a new individual. Induced Pluripotent Stem Cells (iPSCs), have that capacity. To create bovine iPSCs one must turn off some of the cellular genes and turn on the genes for embryonic development. This year we created a cell culture system that has the potential to turn off genes in bovine skin cells and turn on their embryonic genes. Lysostaphin has been shown to protect against mastitis caused by Staphylococcus aureus in both genetically engineered mice and cattle. To determine if, as the scientific literature suggests, lysozyme from trout enhances lysostaphin's ability to protect against S. aureus infections we have produced genetically engineered mice expressing trout lysozyme in their milk and have developed a laboratory test to determine if lysostaphin and trout lysozyme can act synergistically. It is possible to make genetically identical copies (clones) of some individuals but not others. The biological basis for that difference is unknown. It is possible that the proteins that "package" the genes of these two groups differ. To test that hypothesis, we have collected tissues from clonable cows and those that could not be cloned and are examining a number of features (histone modifications and DNA acetylation) associated with their chromosome's structure. Resistance to conventional antibiotics is increasing among mastitis-causing pathogens. We predict that phage endolysins and other peptidoglycan hydrolases that act externally are ideal antimicrobials. To validate this hypothesis we have bioinformatically categorized the predicted staphylolytic enzymes in Genbank into nine unique groups, expressed and purified a representative from each group and characterized their relative staphylolytic activity in vitro and in milk. With deletion analysis we are identifying the active lytic domains. These nine peptidoglycan hydrolase enzymes and others are being tested for synergy against methicillin-resistant Staphylococcus aureus and streptococci. To reduce the risk of resistance development, we have constructed triple acting fusion constructs with three lytic activities in a single protein, and demonstrated that all three lytic domains were active in the final fusion constructs. It is unlikely that a bacterium can develop resistance to three simultaneous lytic activities. These fusions are being tested in vitro, ex vivo in rat blood, for the ability to suppress resistant strain development and for relative efficacy in milk, and animal models of nasal colonization. We have demonstrated that both LysK and lysostaphin SH3b cell wall binding domains can redirect a streptococcal lysin (LambdaSa2) to digest staphylococcal cell wall peptidoglycan, while maintaining high activity toward streptococcal peptidoglycan. Thus we have created dual acting constructs that are lytic for both streptococcal and staphylococcal mastitis pathogens. 4.Accomplishments 1. Protein fusions that kill chronic intracellular S. aureus. S. aureus is a notorious intracellular pathogen causing chronic bovine mastitis. Residence within mammary epithelial cells affords the pathogen a high level of protection from both the host immune defenses and conventional antibiotics, resulting in the routine practice of culling chronically infected dairy cattle. We have demonstrated that the fusion of a peptidoglycan hydrolase enzyme (lysostaphin) to a small (13 amino acid long) protein transduction domain called TAT can carry the lysostaphin enzyme across the plasma membrane of cultured mammary epithelial cells (MAC-T cells) and kill intracellular S. aureus. This effect is not limited to just mammary epithelial cells, but has also been achieved in cultured murine osteoblast cells (bone progenitors). This work has resulted in a provisional patent application and numerous technology transfer and grant writing efforts geared toward a field trial to test the Lyso-TAT in chronically infected lactating dairy cattle. If the Lyso-TAT fusion is able to cure chronic mastitis, this could result in tremendous saving to the dairy industry by avoiding the current practice to cull chronic staphylococcal infected cattle (8 to 15% of US herd annually). 5.Significant Activities that Support Special Target Populations We are currently collaborating in developing a program to create genetically modified goats. The Animal Science Program of the College of Agriculture of the Fort Valley State University (1890 Institution), has a Meat Technology Center, and a Georgia Small Ruminant Research & Extension Center with about 250 goats of various breeds. Assisted in submission of grant application to CREES for research support for “Production of transgenic goats with enhanced muscle mass by myostatin gene-targeting”. Coauthored an abstract for presentation at the ‘Keystone Symposium on Frontiers in Reproductive Biology and Regulation of Fertility.’ 6.Technology Transfer Number of New CRADAS 1 Number of Active CRADAs 1 Number of Invention Disclosures Submitted 1 Number of Web Sites Managed 1 Review Publications Van Hekken, D.L., Wall, R.J., Somkuti, G.A., Powell, A.M., Tunick, M.H., Tomasula, P.M. 2009. Fate of lysostaphin in milk from individual cows through pasteurization and cheesemaking. Journal of Dairy Science. 92:444-457. Hoopes, J.T., Stark, C.J., Kim, H.A., Sussman, D.J., Donovan, D.M., Nelson, D.C. 2009. A Novel Use of a Bacteriophage Lysin, PlyC, as a Disinfectant against Streptococcus equi. Applied and Environmental Microbiology. 75(5):1388-94. Wall, R.J., Powell, A., Sohn, E.J., Foster-Frey, J., Bannerman, D.D., and Paape, M. 2009. Enhanced host immune recognition of mastitis causing Escherchia Coli in CD-14 transgenic mice. Animal Biotechnology. 20:1-14 Donovan D.M., Foster-Frey J., Garrett W.M., Blomberg, L. 2008. Resolving the database sequence discrepancies for the Staphylococcus aureus bacteriophage phi11 amidase. Journal of Basic Microbiology. 48(1):48-52. Becker, S., Donovan, D.M., Foster-Frye, J. 2008. The phage K lytic enzyme LysK and Lysostaphin act synergistically to kill MRSA. FEMS Microbiology Letter. 287(2):185-91. Donovan, D.M., Foster-Frye,J., 2008. LambdaSa2 prophage endolysin requires Cpl-7 binding domains and Amidase-5 domain for antimicrobial lysis of streptococci. FEMS Microbiology Letter. 287(1):22-23. Becker, S.C., Don, S., Baker, J.R., Foster Frey, J.A., Pritchard, D.G., Donovan, D.M. 2009. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiology Letter. 294(1):52-60. Wang, J., Kim, J., Donovan, D.M., Becker, K.G., Li, M.D. 2009. Significant modulation of mitochondrial electron transport system by nicotine in various rat brain regions. Mitochondrion. 9:186-195. Becker, S.C., Foster-Frey, J., Stodola, A.J., Anacker, D., Donovan, D.M., 2009. Differentially conserved staphylococcal SH3b_5 cell wall binding domains confer identical staphylolytic activity to a streptococcal prophage endolysin lytic domain. FEMS Immunology and Medical Microbiology. 443(1-2):32-41.