TRANSMITOCHONDRIAL LIVESTOCK

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0225853
• Project No.: ALA052-1-11012
• Project Start Date: Oct 1, 2011
• Project End Date: Sep 30, 2013

Project Director
Pinkert, C.

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
Pathobiology (Vet Med)

Non Technical Summary
This project will facilitate development of animal genetic engineering efforts at Auburn University with potential for generating new animal systems of commercial value. Development of value-added production traits is envisioned; of benefit to swine and cattle producers, with a possible spin-off of biomedical relevance for the health and well-being of Alabamians. Collaborative studies would be fostered by preliminary data established in this project. The proposed work will enhance our competitiveness by demonstrating those capabilities necessary to attract federal and industry support.

Animal Health Component

20%

Research Effort Categories

Basic

40%

Applied

30%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3999	1040	30%
304	3999	1040	20%
302	3999	1010	10%
301	3999	1020	10%
308	3999	1050	10%
308	3999	1080	10%
315	3999	1020	10%

Knowledge Area
308 - Improved Animal Products (Before Harvest); 303 - Genetic Improvement of Animals; 302 - Nutrient Utilization in Animals; 301 - Reproductive Performance of Animals; 315 - Animal Welfare/Well-Being and Protection; 304 - Animal Genome;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
1080 - Genetics; 1020 - Physiology; 1010 - Nutrition and metabolism; 1050 - Developmental biology; 1040 - Molecular biology;

Keywords

livestock

transgenesis

mitochondria

mtdna

mitochondrial transfer

cybrid

genetic engineering

cloning

transmitochondria

xenomitochondria

heteroplasmy

stem cells

gene transfer

electron transport chain

Goals / Objectives
The overarching goal of this project is to develop transmitochondrial farm animals with enhanced metabolic traits. Aim 1) Speed the production of a transmitochondrial model at AU that incorporates mitochondrial gene transfer. Initially, we propose to introduce species-specific mitochondria and/or mtDNA into the mitochondrial genome of mice. Aim 2) Interact with producers across the state to identify livestock populations for study, collect samples, and highlight AU technology. Aim 3) With proof-of-principle established in mice, and domestic animal sampling in hand, comparable technology will be developed in domestic farm animals.

Project Methods
In comparison to methods successfully employed for nuclear gene transgenesis in livestock over the past 25 years, the lack of comparable recombination in mtDNA has, until recently, prevented direct in vivo manipulation. We propose to develop and refine methods for modification of the mt genome in pilot mouse studies and to establish comparable technology in farm animals (here beef cattle, dairy cattle and swine). Underlying hypotheses are that: 1) for every species there exists an "optimal" mtDNA genotype; and 2) farm animal mt modification is possible; thus offering opportunities for engineered enhancement of metabolic efficiency. Targeted areas for studies with transmitochondrial farm animals include: a) identifying mt populations from select seed stock to facilitate bioengineering of metabolic/mt gene function to produce animals better suited to US and related global environments; b) improving metabolic efficiency of economically important farm animals; and, as a byproduct of these studies, c) modeling human mt function/dysfunction. The long-range projections for and sustainability of US agriculture and food systems could be dramatically affected by a targeted 10% increase in metabolic capacity of farm animals. The National Pork Producers Council reported nearly 35,000 direct/full-time pig production jobs and 515,000 indirect jobs in 2007. In that year, we produced nearly $22 billion in personal income from sales of more than $97 billion adding $35 billion to the US GNP. Couple such impact with training and broad international opportunities for the next generation of agricultural scientists and the return-on-investment from this project is magnified considerably. This project will facilitate development of genetic engineering and transgenic domestic animal efforts at AU. Collaborative studies would be fostered by preliminary data generated in this project (comparable to how "conventional" pig transgenesis was established in Alabama in the 1990s by our collaborative efforts). This project will emphasize sustainable practices, augmented by biotechnology, that consider a food, fiber and agricultural system beginning at production. Included within this framework will be an educational/outreach emphasis on animal stewardship, niche food and value-added products. Accordingly, this project relates directly to long-range improvements in and sustainability of state, national and global animal agriculture by establishing foundational biotechnological tools needed to produce value-added animals, while providing training opportunities for the next generation of bioagricultural scientists on an international scale. As such, this project specifically addresses and is intricately associated with the priorities associated with the FY2010 AFRI Global Food Security program. Furthermore, this project directly relates to long-range improvements in and sustainability of Alabama animal agriculture by developing value-added animals. Proposed research will enhance our competitiveness by demonstrating capabilities necessary to attract federal and industry support.

Progress 10/01/11 to 09/30/13

Outputs
Target Audience: Target audiences reached included undergraduate, veterinary, graduate and postdoctoral students and trainees. Presentations were made to local, regional and national audiences (both professional and lay audiences) through 2013. Changes/Problems: Project is terminated early because the P.I. has left Auburn University. What opportunities for training and professional development has the project provided? Undergraduate, veterinary/professional, and graduate student training in addition to postdoctoral and visiting scholar training at Auburn University. How have the results been disseminated to communities of interest? Dissmination has focused on presentation atprofessional/research meetings (local, state, national and international), formal Auburn coursework presentations,and to various non-technical audiences as well. Additionally, through publication and database/genbank sequence submissions, we have provided results of our studies in various forums. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We generated a number of transmitochondrial and transgenic mouse models. Disseminationof information related to these models and the research focus was provided to various lay and research constituencies within the state, and at national and international conferences. Pig mitochondrial genomic sequence data were generated and reported toward development of genetically modified domestic farm animals.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Cannon, M.V., M.H. Irwin, M.C. Kohn, T.D. Brandebourg, D. Dikic and C. A. Pinkert. Mitochondrial DNA sequence and phylogenetic evaluation of geographically disparate Sus scrofa breeds. Animal Biotechnology.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Irwin, M.H., K. Parameshwaran and C.A. Pinkert. 2013. Mouse models of mitochondrial complex I dysfunction. Intl. J. Biochem. Cell Biol. 45:34-40.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: At this time, grant applications have been submitted to establish funding for outlined objectives. Additionally sequencing data generated were used in identifying potential mitochondrial modeling targets. Lastly, mouse technologies continue to develop and were reported at scientific meetings. PARTICIPANTS: Carl A. Pinkert, Auburn University; Michael H. Irwin, Auburn University; Frank F. Bartol, Auburn University; Kodeeswaran Parameshwaran, Auburn University; Matthew V. Cannon, Auburn University; David A. Dunn, Auburn University; Michael C. Kohn, Auburn University; Hannah P. Findlay, Auburn University; Kaitlin M. McCarthy, Auburn University; Ian A. Trounce, University of Melbourne, Australia; Kumiko Takeda, ILGS, NARO, Tsukuba, Japan; Terry D. Brandebourg, Auburn University; Kosta Steliou, Boston University; Domagoj Dikic, University of Zagreb, Croatia. TARGET AUDIENCES: Target audiences include constituencies within the state of Alabama and southeastern US, as well as nationally and internationally in relation to food production, sustainable agriculture and livestock commodity groups. Socially, economically, or educationally disadvantaged constituencies are also represented in regard to efficient generation of food and fiber. Our efforts at influencing metabolism in farm animals focuses on engineered cellular function (mitochondrial biology) to achieve desirable metabolic traits in terms of feed efficiency, optimal lipid metabolism and better meat quality (high protein and healthy fat content), along with improved sustainability under diverse and often challenging regional environmental conditions. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This is a work in progress and preliminary in nature. Additional outcomes and impacts are anticipated in the next reporting period. In current studies, next generation sequencing of mitochondrial DNA (mtDNA) targeted our understanding of breed derivation and metabolic characteristics of production animals and traits. Sequence analysis of mtDNA from purebred swine with highly disparate production characteristics was initiated to evaluate mtDNA haplotype diversity as a possible influence on mitochondrial function. Differences in mtDNA sequences between breeds suggest function-altering changes in protein sequences underlying differences in metabolic traits between swine breeds. The sequence data will form the basis of future studies into the role of mitochondrial function in determination of production traits in domestic swine. Lastly, such studies may provide insight into how mitochondrial function and mtDNA haplotype would influence obesity.

Publications

• Dunn, D.A., Cannon, M.V., Irwin, M.H., and Pinkert, C.A. (2012). Animal models of human mitochondrial DNA mutations. Biochim. Biophys. Acta 1820:601-7.
• Takeda, K., Tasai, M., Iwamoto, M., Oe, M., Chikuni, K., Nakamura, Y., Tagami, T., Nirasawa, K., Hanada, H., Pinkert, C.A., and Onishi, A. (2012). Comparative proteomic analysis of liver mitochondrial proteins derived from cloned adult pigs reconstructed with Meishan pig fibroblast cells and European pig enucleated oocytes. J. Reprod. Dev. 58:248-253.
• Shi, J., Irwin, M.H., and Pinkert, C.A. (2012). Liposome-mediated transfer of mitochondria harboring foreign mitochondrial DNA into cultured fibroblasts. Auburn Univ. J. Undergrad. Scholarship (AUJUS). 1:8-11.
• Takeda, K., Srirattana, K., Matsukawa, K., Akagi, S., Kaneda, M., Tasai, M., Nirasawa, M.K., Pinkert, C.A., Parnpai R., and Nagai, T. (2012). Influence of intergeneric/interspecies mitochondrial injection; parthenogenetic development of bovine oocytes after injection of mitochondria derived from somatic cells. J. Reprod. Dev. 58:323-9.
• Kohn, M.C., Irwin, M.H., and Pinkert, C.A. (2012). Strategies for modeling mitochondrial metabolic disorders in mice. Boshell Diabetes and Metabolic Disease Research Symp. Auburn AL, #P17, p. 49, March 2.
• Pinkert, C.A., Parameshwaran, K., Cannon, M.V., Dunn, D.A., Kohn, M.C., Steliou, K., Takeda, K., Trounce, I.A., and Irwin, M.H.. (2012). Murine modeling of human mitochondrial disease pathogenesis. Keystone Conf., Banff, Canada, #303, p. 103, March 19-24.
• Kohn, M.C., Irwin, M.H., and Pinkert, C.A. (2012). A novel strategy for targeted mutation of mitochondrial DNA. 22nd Annual Graduate Scholars Forum, Auburn AL, Session 12, Feb. 28-March 1.
• McCarthy, K.M., Irwin, M.H., and Pinkert, C.A. (2012). Localization of recA transgene product in mitochondria of transgenic mice. ResearchWeek 2012: Undergraduate Forum, Auburn AL, #U26, p. UP-14, April 2-5.
• Irwin, M.H., Cannon, M.V., and Pinkert, C.A. (2012). A novel strategy for targeted mutation of mitochondrial DNA. ResearchWeek 2012: Faculty Forum, Auburn AL, p. FO-1, April 2-5.
• Pinkert, C.A., Parameshwaran, K., Cannon, M.V., Dunn, D.A., Kohn, M.C., Findlay, H.P., Steliou, K., Takeda, K., Trounce, I.A., and Irwin, M.H.. (2012). Mitochondrial modeling of mammalian development and human disease. 3rd World Congress, Targeting Mitochondria Conf., Berlin, p. 34, Nov. 8-9.