Christopher K. Tuggle - US grants

A grant has been awarded to Iowa State University to acquire instrumentation for the analysis of DNA oligonucleotide microarrays that allow the parallel evaluation of up to 22,500 genes in a single experiment. This system will enable researchers at ISU to employ state-of-the-art genomics technology to a variety of plant, animal, and human research topics including; the molecular characterization of genes coincident with disease, response to biotic or abiotic stresses, or cellular development.

Oligonucleotide-based microarrays can already be purchased which have been designed for genome analysis of a number of different species. Research programs at Iowa State University that will be most advantaged by this system are those investigating questions in development, metabolic networking, biotic and abiotic stresses, and that involve Arabidopsis, Triticeae (barley/wheat), yeast, Drosophila, C. elegans, mouse, rat, cattle, or human microarrays. A typical experiment could involve an organism infected with a pathogen compared to a control sample that was not infected. Messenger RNA would be extracted from both samples and then used to probe the oligonucleotide microarrays. This RNA contains the genetic code for the specific genes that are active during pathogen infection. The new instrumentation facilitates highly quantitative assessment of the expression levels of each of the 22,500 genes in the two samples, thus, comparison of the two sets of data would point out genes that are correlated with disease responses.

The use of this technology will make it possible to systematically study how genes contribute to complex phenotypes ranging from disease, response to external stimuli, and normal growth and development. The rapid discovery of new genes associated with such complex traits is expected to lead to novel discoveries in medicine and agriculture. Another mission of ISU is to train graduate students and undergraduate students in contemporary biology, and it is essential that they become proficient in the theory and use of modern genomics tools. This instrument will be made available to graduate students conducting research in the departments of Agronomy, Animal Science, Biochemistry, Botany, Plant Pathology, and Zoology & Genetics as well as in the interdepartmental graduate programs in Genetics, Plant Physiology, Ecology & Evolutionary Biology, and Molecular, Cellular, & Developmental Biology. In addition, several students from a NSF-funded REU (Research Experience for Undergraduates, Undergraduate Research Experience in Molecular Biotechnology and Genomics) program will have access to this advanced technology each summer. The REU program focuses on students from traditional minority universities and students from small Midwestern liberal arts colleges. Half of the students are members of under-represented groups and half are women. Thus, the instrumentation system will be integrated into both the graduate and undergraduate education programs at ISU.

This grant will support a symposium entitled "Integration of Structural and Functional Genomics" at Iowa State University (ISU) to be held on 22-25 September, 2005. The symposium is part of a series of symposia held over the past 10 years at ISU in the areas of Growth Factor and Signal Transduction. The purpose of the symposium is to gather together a wide variety of researchers involved in investigation of genome structure/function using molecular and computational biology tools, and statistical methods. With recent, rapid advancements in genome sequences of many vertebrates, the time is appropriate for more broad comparative approaches in the study of genome structure and function. The PIs have recruited 18 outstanding researchers in functional genomic analyses for the plenary addresses including four well-regarded female investigators. This symposium will open up discussion of current research in this field and will help to focus attention on important new directions for future research. There will be plenary talks, a poster session for associated work to be presented and time allotted for informal discussion sessions and workshops. A significant effort is outlined to include participation of students from under-represented groups. The university will use its connections with New Mexico State University and with the Minorities in Agriculture, Natural Resources and Related Sciences (MANRRS) to ensure minority student involvement. Travel grants funded in part by this award will be available to students and junior faculty with special efforts made to support applicants from under-represented groups. Funds from this grant also will be used to defray travel costs for invited speakers.

The main objectives of this proposal are to develop improved statistical methods for detecting sets of genes that are differentially expressed across two or more conditions and to apply these methods to discover new genetic and physiological mechanisms that control food intake, nutrient utilization, energy regulation, and metabolism. The developed statistical methods will provide powerful alternatives to tests of enrichment
or overrepresentation that have become popular tools for interpreting microarray experiments. The proposed methods gain advantages over existing methods by (1) recognizing, accounting for, and utilizing dependence among genes; (2) maintaining continuous information about the degree of difference between gene set expression distributions; (3) identifying interesting gene sets by comparison of gene sets across treatments rather than comparing gene sets to one another; and (4) capturing information about differential expression contained in the joint expression distributions rather than using only marginal distributions.
In addition to their use for identifying differentially expressed gene sets in traditional microarray experiments, the proposed methods offer a new and powerful approach for identifying genetic loci that control the expression of gene networks. The integrated research team of statisticians and biologists will identify the best of the proposed methods by theoretical study of their asymptotic properties, by comparisons of their performance on simulated data sets designed to mimic structures found in real data sets, and by weighing the value of biological insights provided by their application to actual data from a variety of microarray experiments. The asymptotic framework used in this research considers the statistical properties of the testing procedures as both the dimension of the data vectors (number of genes in a set)and the sample size (number of experimental units) grow large. Such a framework permits evaluation of methods for use on data of very high dimension and produces results that are intrinsically interesting from the statistical point of view.

The developed methods will be used to investigate genetic control of food intake and energy regulation in pigs and to discover genetic regions that control the expression of gene networks in a population of mice that serve as a model for human obesity. The insights provided by these studies may be used to develop treatment strategies for human obesity. In addition, the proposed methods have much broader application to nearly any microarray-based investigation of differential gene expression. Applications range from the identification of sets of genes that play a role in distinguishing cancerous tissue from non-cancerous tissue to the identification of sets of genes important for developing high-quality plant material suitable for conversion to biofuel. The general goal of this work is to provide scientific researchers with powerful tools for identifying the most important genes behind a wide variety of biological phenomena.

An international symposium, Systems Biology: Integrative, Comparative, and Multi-Scale Modeling, will be held at Iowa State University, June 11 - 14, 2009. The Symposium will emphasize integrative and comparative systems biology, with sessions on modeling in contexts that include genomics and network control of transcription, protein interactions and metabolism in plants, animals and bacteria. The integration of biology, computation and technology is critical for further advances in understanding complex biological processes such as organismal physiology, ecosystem sustainability and biomass and livestock production. The Symposium is organized to maximize communication at both formal (plenary talks) and informal (poster sessions, roundtable discussions, social events) levels for speakers and Symposium attendees. Broader impacts include a session for scientists and students to examine multidisciplinary educational issues related to training in bioinformatics. Participants will evaluate types of bioinformatics training being used to educate undergraduate and graduate students at small colleges, doctoral granting institutions, as well as through peer-to-peer interactions and on-line learning. The majority of the budget will be used to fund travel grants for students and postdoctoral associates, with an emphasis on encouraging members of under-represented minority groups to participate.

DESCRIPTION (provided by applicant): The promise of stem cell-based therapies is currently not being fully realized due to the lack of appropriate pre-clinical animal models. Consequently, there remains a critical need to develop better animal models that better translate results from preclinical research trials to the patient. Scientific advancements addressing this problem have been realized through the identification of a naturally occurring Severe Combined Immuno- Deficient (SCID) line of pigs. The SCID pig model has been preliminarily characterized as both T and B lymphocytes deficient and thus is deficient in adaptive immunity. The pig is known to be an excellent model for human biology due to similarities in size, physiology and genetic code; and this novel SCID model has been successfully engrafted with multiple human cancer cell lines, indicating high potential as an in vivo model for many areas of regenerative medicine testing. We have received multiple requests and interest in obtaining this model. The research areas of these interested scientists spans many areas of regenerative medicine research, from cardiac progenitor cell therapies to cartilage regeneration studies, to the use of mesenchymal stem cells for bone regeneration and wound repair, to improving treatment of graft versus host disease in hematopoietic stem cell (HSC) transplants. A pig SCID model will have a specific positive impact in that it will provide researchers with alternative and comparative models for such stem cell research. Our long-term goal is to create validated SCID models for broad use in preclinical testing of such stem cell based therapies. The specific objectives of this application are to further develop this model for regenerative medicine testing, as well as improve its ability to engraft human cells into the bone marrow so that a human immune system can develop. Such an advanced model could be broadly used for vaccine testing and the study of human-specific pathogens. To maximize the broad use of these models, we will further develop protocols and design and test biocontainment facilities for practical utilization of our current pi lines. Our rationale for the proposed research is that the SCID pig is physiologically and phylogenetically more similar to humans than the mouse and therefore may more accurately reflect how proposed stem cells will survive and function in humans. This project is innovative because a SCID pig model has not been fully developed, and development of such model may result in significantly and rapidly advancing the fundamental knowledge of SCID into translational medicine capable of accelerating regenerative medicine research. With regard to expected outcomes, the successful completion of this project will create multiple genetic resources and associated animal procedures that will be highly desirable for SCID based modeling for research projects focused on the efficacy of stem cell therapeutics. Thus these unique resources are expected to have a significant impact in accelerating the translation of regenerative medicine research into the clinic.

Biology is transitioning from a descriptive science to a predictive science, where rules for outcomes (phenotypes) given specific genomic starting points (genotypes) can be developed and tested. The biological challenge is to understand the instructions encoded in genomes and to predict the resulting phenotypes, i.e., the "genotype to phenotype" question. A nascent project named the Functional Annotation of ANimal Genomes (FAANG) with over 300 members has been developed at the grassroots level to initiate annotation of domesticated animal genomes and eventually the genomes of other species (www.faang.org) because these experimental systems offer great potential to address the genotype to phenotype question. This award partially supports a workshop to discuss the coordination of the FAANG initiative. This Gathering On FAANG (GO-FAANG) Workshop will have an emphasis on small and large group discussions designed to develop policies and practical approaches to maximize the probabilities of success of this crucial next phase in animal genomics. Participation of both academic and government scientists at various stages of their career will be encouraged by providing diverse opportunities for interaction in small and large group settings.

A slate of diverse speakers has been developed that will provide a glimpse into the incredible opportunities available in animal functional genomics as well as a perspective on integrating such vision with the status of the FAANG project. This approach will provide clarity as to the specific needs for success of FAANG. Community discussions will be focused on accomplishing the initial goals of FAANG, which include (a) establishing priorities for research efforts; (b) planning the management structures required for efficient use and sharing of samples, data and computational tools; and (c) identifying resources needed to accomplish these goals. Outcomes from the workshop will be published in peer-reviewed journals and posted for public dissemination on the FAANG www-site.

Project Summary/Abstract Large animal models are critically needed to develop regenerative medicine therapeutics and maximize safe translation to the clinic, as the FDA recommends data be provided on both small and large animal models for approval of such therapies. Using R24 funding (OD019813), we developed biocontainment facilities and protocols to raise an Artemis (DCLRE1C) mutant SCID pig model (ART) as well as an improved model also mutant in IL2RG (ART-IL2RG). The use of SCID pig models in preclinical research necessitates the ability to safely ship healthy SCID pigs across the United States to interested scientists who do not have the ability to raise such pigs themselves. The following two Aims address this existing deficit in the SCID pig research field: Aim 1. Create methods to safely transport SCID pigs within a HEPA filtered isolator transported in a HEPA filtered trailer to researchers in the US and Canada Aim 2. Improve the biosecurity and health of SCID pigs by creating a shower-in facility and purchase dedicated sterilization equipment. As there is no established method to ship SCID pigs while maintaining biocontainment, work in Aim 1 will focus on development of a transportation system consisting of a customized trailer and internal isolators will make this critical next step possible. The transportable internal isolators are needed both to safely house the SCID pigs as well as short- distance transportation from facility biocontainment bubbles to the sterilized trailer for long-distance transport. Further, this revised plan expands the type of clientele who can use the SCID pig, as the isolators containing the SCID pig can be maintained at an external site for short-term research studies. In Aim 2, we continue to develop effective protocols for SCID pig rearing and have identified that isolators are a useful adjunct to the originally proposed and validated bubble procedures. Biosecurity of the SCID pigs will be dramatically improved by the addition of a shower-in facility and purchase of dedicated hydrogen peroxide vaporizer adequately sized for sterilizing these new spaces and equipment. We propose to use A&R funds to renovate unused space adjacent to the existing biocontainment bubbles to provide this shower-in facility and raise the level of biosecurity for SCID pig housing to the industry standard. The sterilization equipment, consisting of a dedicated hydrogen peroxide vaporizer capable of handling the existing bubbles and the proposed new transportation approach in Aim 1, is critically needed as existing equipment is inadequate. The proposed Supplemental Aims do not expand the scope of the existing parent grant and are directly responsive to the call for A&R supplements.