Barbara B. Knowles - US grants

Tumors result when cells with defects in proliferation control arise that are indistinguishable from normal cells by the host's immunocytes. We are investigating the expression of the epidermal growth factor receptor (EGFR) in normal and tumor cells as well as the host's immune response to viral transforming proteins. Normal human fibroblasts progress with passage in vitro from EGF responsive (young) cells into a nonproliferating senescent state. By comparative analysis of the EGFR from young and senescent cells, we find that although it is equivalently expressed by both cell types and binds comparable levels of EGF, the autophosphorylating tyrosine kinase activity of EGFR is defective in senescent cells. This defect provides a mechanism for removal of normal cells from the proliferating pool and we are investigating how this aberration arises. The stem cells of teratocarcinoma share properties with those of the implanting embryos and like embryonic cells, can be induced to differentiate in vitro into a variety of cell types. We have found human teratocarcinoma stem cells express EGFR and their differentiated derivatives do not. Such a change results in cell populations in loco with differing requirements for cell growth control and suggests a mechanism of development based on expression of cell surface receptors and availability of growth factors. While investigating the relationship between hepatitis B virus integration, chromosome rearrangements, and abnormal growth control in human liver tumors, we found one hepatoma cell line that contains a rearrangement of the region of the chromosome that encodes EGFR, and expresses 20-fold more EGFR than other hepatoma cells. Thus, genetic rearrangements can lead to defective growth control through increased expression of a normal growth factor receptor. We find that strong protection against SV40-induced tumors in the mouse is correlated with the host's ability to mount a cytotoxic T-cell immune response to the viral transforming protein, SV40 T-antigen. SV40 transgenic mice (obtained by injecting SV40 plasmids into the zygotes) that die of progressive SV40 tumors are immunologically tolerant to SV40 T-antigen while transgenics (containing the same plasmid) that remain tumor-free are not. We hypothesize that the stage of development at which the SV40 T-antigen is expressed determines if it is immunologically recognized as foreign and rejected, or as self and tumors develop. (A)

Chronic infection with hepatitis B virus (HBV) has been linked by extensive epidemiological evidence with the incidence of hepatocellular carcinoma (HCC) in man. HBV sequences are found integrated in host cellular DNA from tumor biopsies and from several HCC-derived cell lines, including Hep 3B (containing one copy of HBV) and PLC/PRF/5 (containing 6-8 copies). Both cell lines synthesize the HBV surface antigen and synthesis appears to be regulated according to the growth state of the cells. The proposed research will investigate the integrated state of HBV in HCC cell lines, and in particular in the Hep 3B line, whose single integration site will greatly simplify analysis of the relationship to HCC. The HBV integration sites in this and other cell lines will be chromosomally mapped to determine if integration occurs on a common chromosome and if there is correlation with karyotypic abnormalities seen in HCC lines. The Hep 3B integration site will also be isolated by genomic cloning to determine the integrated configuration of viral sequences. Flanking host sequences from the genomic clone will be used as probes of Southern blots to study how integration occurred and Northern blots to determine whether any host transcription is affected. The proposed research will also study gene expression by the integrated HBV sequences. Potential regulatory mechanisms will be examined by DNase I sensitivity and methylation analysis, and these results will be examined by DNase I sensitivity and methylation analysis, and these results will be compared in cells grown under conditions known to alter HBsAg expression. Transfection experiments will then be undertaken to examine whether this regulation is controlled by native viral sequences or if an integrated configuration is required. A transfection assay will also be used to precisely map the HBsAg promoter, with particular attention to the possibility of regulatory sequences. These results, along with a detailed analysis of HBV in its integrated form, should increase our understanding of the mode of action of this potentially oncogenic virus.

To determine the control of the differentiated functions of the B- islet cells of the pancreas and to investigate the autoimmune etiology of insulin-dependent diabetes, well-characterized cell lines of defined genetic background are necessary. We have derived and characterized one such cell line from an insulinoma which arose in an SV40 T-antigen transgenic mouse. Based on our preliminary studies of the control of insulin release by these cells, we propose to address the interactions between nutrient and hormone that mediate insulin release. The IgSV195 cell line provides a particularly useful model to study the events which may lead to immune recognition of islet cell antigens. IgSV195 is derived from a tumor which arose in a genetically defined mouse strain, and these cells express SV40 T-antigen, a potent inducer of humoral and cellular immunity in the mouse. We propose to determine whether this antigen is spontaneously recognized by syngeneic mice bearing IgSV195 insulinomas or whether infection with the M strain of a EMC virus can induce its recognition by host lymphocytes. Finally, since the success of transfection experiments requires recipient cell line(s) of the same state of differentiation and preferably of the same species of origin as the genes to be tested, we plan to derive a cell line(s) from biopsies of human insulinomas. Each of these projects are designed as pilot studies to increase our expertise in the area, to accumulate data preparatory to a full grant application, and to determine the feasibility and relevance of these approaches to the study of diabetes.

Differentiation of arrays of cells in the intact organism is accomplished by sequential display of specific molecules capable of transmitting information about their surroundings through intercellular intermediates to effect a change in the cell's genetic program. To approach the role of intercellular contact in determining differentiation, we propose to develop an in vitro system capable of mimicking cell-cell interactions, specifically between prothymocytes and thymic stroma cells. In this system, intimate cellular contact between the thymic stroma and the prothymocyte probably plays a major role in the initiation of differentiation of the prothymocyte into the major T-cell lineages. To test this hypothesis we propose to derive in vitro functional correlates of each cell type and to this end we will: A. Characterize the morphologically distinct stromal cell types we have clonally derived in vitro from the hyperplastic thymus of an SV40 transgenic mouse and determine the interactions that lead to proliferation and differentiation of the prothymocyte. B. Derive in vitro stable populations of replicating prothymocytes and determine if they are capable of differentiation by means of interaction with the thymic stromal cell lines.

The relationship between HBV infection and the induction of hepatocellular carcinoma is currently enigmatic. We will explore several mechanisms that might account for association of HBV infection and liver tumor formation. We have observed instability in the genome of the host presenting with hepatocellular carcinoma and in chronic carriers of HBV. We therefore propose to explore the possibilities that HBV might integrate in specific areas of the host genome predisposed to instability that HBV might induce such genome destablization or that environmental factors or hereditary factors are at the root of the instability observed. Further, we propose to determine if genome instability is of predictive value in these tumors. We have established a panel of hepatocellular carcinoma-derived cell lines of hepatocyte morphology and function and plan to continue our analysis of their common chromosome aberrations. We propose to determine specific breakpoints common to these cell lines to identify specific areas of the genome which may contain genes responsible for the control of hepatocyte proliferation. By analysis of somatic cell hybrids, derived from fusion of human hepatocellular carcinoma-derived cell lines and stationary cultures of rat hepatocytes, and transfection of the stationary rat hepatocyte with cloned HBV and human hepatocellular carcinoma-derived DNA, we plan to distinguish between the direct oncogenic potenital of HBV and that of potentially activated human genes. Furthermore, identifiecation of a gene(s) of a gene product(s) that might effect such transformation is proposed.

The overall goal of the Program Project is to elucidate the complex interactions between the cellular and genetic control mechanisms which regulate the early stages of mammalian development. During the series of cleavage division which leads from zygote to blastocyst, sequential activation of the embryonic genome occurs concomitantly with the first overt signs of differentiation of trophectoderm and the origins of embryonic tissues. By the blastocyst stage, the embryo contains the lineage precursors of all embryonic and extraembryonic tissues. To determine the nature of these early and crucial differentiation events, we propose the following experimental approaches: 1) Through the joint effort of all investigators, genetic probes which reflect the gene expression at each developmental stage will be developed. These probes will be used to identify the corresponding gene products and to begin the analysis of their function. 2) By transient or permanent introduction into embryos of specific genes and/or their products, the functional role of these genes will be determined. 3) Nuclear transfer embryos will be analysed to determine the reasons for their developmental failure and for identifying the genes involved. Part of this approach will involve analysis of specific and gene regulation during gametogenesis. 4) The role of growth controlling molecules (including growth factors and their receptors and oncogenes) in regulating the growth and differentiation patterns in early embryos including trophectoderm and inner cell mass will be studied. The role of protein phosphorylation in regulating the transcriptional activity of the embryonic gene will be examined. 5) Establishment of cell lineages and the cell lineage specific gene expression will be explored by injection of suitable DNA markers and by in situ hybridization with specific probes. 6) Differentiation of trophectoderm, trophoblast and inner cell mass will be studied by identifying genes and gene products specific for those tissues, by determining their role and by attempting to specifically alter or abolish this first differentiation event in mouse embryogenesis. In view of the accessibility of trophectoderm tissue, the readily characterized sequence of developmental events involved in trophectoderm formation.

The genetic changes which lead to development of primary hepatocellular carcinoma (PHC) remain enigmatic, although several possible etiologic agents have been identified. The major goal of this proposal is to investigate the genetic changes common to PHC of different etiologies in an effort to understand development of this disease. Our previous investigations pointed to consistent translocations and deletions of chromosome 1 in hepatoma-derived cells. Comparison of genomic DNA obtained from tumor cells and normal cells from the same patient using "RFLP probes" which map to distal 1p revealed loss of genetic heterozygosity in the tumor cell. On the basis of these preliminary data, we hypothesize that the loss of a tumor suppressor gene is a likely and possibly initiating step in PHC. Our proposal is geared to substantiating this chromosome 1-encoded genetic change in PHCs of multiple origins and to defining how this and the genetic changes described by others may lead to PHC. To accomplish this, we will look for genetic instability and chromosome loss in premalignant livers and will transfer individual normal human chromosomes into different hepatoma-derived cell lines to determine if they suppress tumor cell growth or tumorigenicity. To verify our hypothesis of the importance of 1p loss of heterozygosity, we will extend our study to tissue from more patients and to other probes in order to pinpoint the region containing the putative suppressor gene, a prerequisite for its cloning.

DESCRIPTION (Adapted from applicants description): The molecular mechanisms that govern the earliest stages of mammalian preimplantation development remain largely unexplored because, until recently, there were no molecular probes to investigate this process. The mammalian egg is transcriptionally inert, and the embryonic genome is not immediately transcribed upon fertilization. Consequently, key development processes during this period are governed by timely translation of stored maternal transcripts and cascades of post-translational modifications. The investigators have devised a strategy to determine the function of maternal transcripts during early preimplantation development. The Cre/loxP conditional null mutagenesis where Cre-induced disruption of the genes under investigation is confined to the oocyte by the controlling sequences of the zona pellucida 3 gene. Mutation eliminates the transcription of the targeted gene in the growing oocyte so there is no maternal mRNA in the embryo. The first gene we propose to target is the cell adhesion molecule E-cadherin that mediates embryonic compaction. Previous classical knock-out studies of E-cadherin have demonstrated that the homozygous null mutants die at the time of implantation. However, the embryo compacts and survives to from the blastocyst. If E-cadherine is essential to the initiation of compaction, then it is likely synthesized from residual maternal mRNAs. Since the mutation is an embryonic lethal, the effect of the lack of its maternal mRNA could not be addressed in the offspring. An oocyte-specific knock-out experiment to study the role of maternally encodes E-cadherin in compaction will be performed.

DESCRIPTION (Applicant's Description): Researchers are increasingly using new methods for controlling gene expression in whole animals to investigate the regulation of normal mammary gland development and neoplasia. The purpose of this meeting is to conduct the first comprehensive discussion about the use of new molecular genetic mouse models for understanding the mechanisms operative in mammary gland morphogenesis, differentiation, involution, and neoplasia. Opportunities for gaining insight into mechanisms of human beast cancer from these models will be a central focus. The specific objectives are to (1) communicate recent advances in understanding the molecular and cellular mechanisms controlling normal mammary development; (2) delineate current hypotheses regarding oncogenesis in the mammary gland, including molecular pathways, modes of activation, and processes of cellular transformation and disease progression; (3) define the utility of the mouse for modeling human breast disease through discussion on mouse strains carrying targeted mutations in human susceptibility genes and comparative pathobiology; (4) explain emerging technologies for targeting gene expression to the mammary gland at specific times; (5) discuss the resources available to the community and determine whether additional resources are needed; (6) define promising areas for new research through discussions on the pressing questions about molecular mechanisms in human breast cancer that can be answered using mouse models; and (7) compile a Proceedings to be published on the World Wide Web that will serve as a timely resource for investigators interested in learning about the mouse as a model for breast cancer research.

Analysis of gene function at the molecular. cellular and organismic level requires the availability of diverse sophisticated analytic tools. The Jackson Laboratory (TJL) is making major contributions in the area of mammalian genetics with active research programs and vast resources of mouse models and bio-informatic databases. The success of a majority of these programs relies heavily on state-of-the-art equipment to facilitate imaging at finer and more quantitative levels to follow the minute changes a single gene may cause. It is critical that the research programs of TJL have access to this level of technology to allow expansion of the research to new levels of resolution and therefore, understanding. The Jackson Laboratory proposes to incorporate the requested Leica TCS 4D Confocal Microscopy System integrated with the Leica DMRE Upright Microscope, into a network of research and resource programs aimed at developing and analyzing mouse models of human diseases. Presently, there is no similar state-of-the-art system available to researchers at TJL or in this area of the state. Researchers at TJL desperately need access to a state-of-the-art confocal microscope and the ability to render those images as 3D reconstructions. The currently available. mercury-source fluorescence microscope, is inadequate to specifically localize fluorescent probes at the required resolution, and render the data in a format providing the necessary dimensional information. The equipment requested in this application was selected by an active scientific advisory committee specifically to encompass all the current and foreseeable needs of the Scientific Staff at TJL that are not able to be met through traditional funding mechanisms. It will be maintained and utilized to the limits of its technology in a core of centralized Shared Scientific Services that have produced quality research at TJL for over 30 years. The system requested will expand the boundaries currently set by the limitations of our current technology, and bring us into the twenty-first century.

Breast cancer is a tragedy affecting nearly 180,000 newly diagnosed women and killing more than 43,000 each year. One of the requirements for progress in understanding the etiology and improving the treatment of this disease is the development of new animal models in which normal mammary gland biology and all stages of carcinogenesis can be investigated. The tools are in place to prepare new mouse models for mammary gland research; what is needed is a dialog between basic and clinical researchers about the molecular and tumor biology of human cancer initiation and progression and their correlates and approaches to modeling in mice. The Jackson Laboratory is establishing a series of meetings on mouse models for human cancers with the overall goal of discussing the development and validation of accurate preclinical mouse models and their use in translational cancer research. The first meeting, (October 5-8, 1999) will focus on mammary carcinogenesis with the specific objectives of: l) evaluating current approaches to modeling aspects of human mammary cancer in mice; 2) delineating current hypotheses regarding oncogenesis in the mammary gland; 3) communicating recent advances in understanding and modeling processes of angiogenesis and metastasis; 4) comparing and contrasting the hormonal control of mammary epithelium in human and mouse; 5) discussing novel therapeutic and preventative strategies and mouse models needed to test them; 6) providing experience in interpreting and comparing histopathology in mouse and human; 7) discussing informational and other resources needed by the community; and 8) preparing an online proceedings of the meeting.

Cancer is a genetically complex and biologically heterogeneous group of disorders. It has become increasingly clear that the - laboratory mouse, the best genetically-defined experimental model organism for humans, presents a major opportunity for rapid advancement in understanding the genetic basis of cancer. Therefore, a broad-based training in mouse genetics, genomics and biology is critical to meet this end. The overall goal for our course is to train young scientists (predoctoral, postdoctoral trainees, new investigators) and to retrain established investigators in the use of genetically-defined laboratory mice as genetic tools for asking questions about gene function. Students completing the course will acquire working and critical knowledge of experimental approaches to: (1) Mouse genetics and genomics, (2) Mammalian development, (3) Mouse models for human diseases, (4) Ethical perspectives of genetic research. These Aims will be accomplished by offering an intensive 2-week course to 30 students chosen for their outstanding research potential. They will interact with a group of prominent mouse geneticists and biologists both from The Jackson Laboratory and other institutions. Student enrollment is kept deliberately small to achieve the desired high level of student-faculty interaction. The course is held in a retreat setting at Highseas, a Jackson Laboratory oceanfront conference center that is the site both for the course presentations and residential accommodations for the students and faculty. Lectures, workshops, and demonstrations are held mornings, afternoons, and evenings for a total of 87 hours of formal instruction. The Jackson Laboratory is an NCI-designated Basic Research Cancer Center and has a long history of presenting advanced courses and scientific meetings including the renowned "Short Course" co-organized with Johns Hopkins University and held here continuously for 37 years. The course on Experimental Genetics of the Laboratory Mouse described in this application has been held annually since 1992. The Principal Investigator and the Organizing Committee collectively have extensive experience in organizing and lecturing in Jackson Laboratory-sponsored and other courses and meetings.

Mouse models with heritable cancers that are accurate, reproducible models of human cancers are urgently needed to advance research into the etiology, treatment, and prevention of human cancers. To exploit these models fully, investigations must be multi-disciplinary, drawing on the expertise of researchers in diverse fields. Understanding of mouse biology and pathology are essential, however, investigators using other model systems or who were trained in other research disciplines bring equally important insights. To achieve a cross-disciplinary, translational research focus on cancer control and prevention, opportunities must be provided for young investigators to have hands-on training in the techniques for preparing and analyzing mouse models of human cancers. To address this need The Jackson Laboratory (TJL) will offer an annual workshop series on mouse models of specific human cancers; the first will focus on mouse models for mammary cancer. The topics of subsequent workshops will be determined after consultation with the NCI Mouse Models of Human Cancers Consortium Steering Committee, TJL faculty, and other experts in cancer research. Faculty will be drawn from TJL staff and invited experts; sessions will include both lectures and hands-on training. Participants will be at the late-postdoctoral/early independent researcher level and will be competitively selected. The specific objectives of the first workshop are to: 1) demonstrate basic animal husbandry techniques needed to propagate and maintain genetically-defined mutant strains at high health levels; 2) demonstrate techniques for analyzing mammary gland anatomy throughout the normal reproductive cycle and during carcinogenesis; 3) demonstrate techniques for in vivo and in vitro manipulation of mammary gland growth; 4) introduce novel technologies for analyzing mammary gland function and neoplastic progression.

Although a clamor arose over the cloning of mammals, the fact that very little is known about how normal mammalian development initiates has gone unrecognized. We have developed a multi-faceted approach to define the molecular control of this time in development and stand ready to address this issue. We prepared a large and representative cDNA library from the 2-cell mouse embryo that is being sequenced by the mouse EST project. Our analysis suggests these cDNAs are, in the main, from maternal transcripts that are targeted for timely translation during the oocyte to embryo transition. The principle that maternal transcripts govern the physiology of the newly formed embryo is derived from studying organisms with readily accessible oocytes and embryos. However, these organisms produce mosaic eggs and their embryos become motile within hours of fertilization, whereas mammalian eggs are nonpolar and their embryos implant in the uterus several days after fertilization. In Xenopus maternal transcripts, UA-rich sequences located at specific sites in the 3'-UTR, determine when they are translated. One of our objectives is to understand how temporal translation of maternal transcripts is controlled in mammals. We find similar UA-rich sequences, albeit with different spatial constraints, may control translation of mammalian maternal transcripts. This knowledge is of predictive value. The presence of such a sequence within a given region of the 3'-UTR suggests when its protein will be biosynthesized. We plan to: 1) define the cis-sequences and siting requirements for transcript translation in the mouse oocyte and embryo; and 2) determine whether biosynthesis of the encoded proteins follows the predicted pattern. Our second objective is to understand the functional role and critical nature of the just-in-time availability of specific molecules. We plan to make oocyte- and stage-specific mutations of two genes functioning within biochemical pathways known to be active in the mouse oocyte and embryo. Our goal is to produce a clear picture of the molecules and pathways necessary for embryogenesis in mammals. In the long range, the understanding gained from this study may lead to new therapies for infertility/fertility and diseases of aberrant progression through the cell cycle, such as cancer.

Colorectal cancer is the second leading cause of cancer deaths in the United States. In 1995, there were about 160,000 new cases of colorectal cancers with 60,000 deaths resulting from the disease. One of the requirements for progress in understanding the etiology and improving the treatment of this disease is the development of new animal models in which normal tissue biology and all stages of carcinogenesis can be investigated. The tools are in place to prepare new mouse models for cancer research; what is needed is a dialog between basic and clinical researchers about the molecular biology of human tumors, their initiation and progression with respect to biological correlates and modeling in the laboratory mouse. To this end, The Jackson Laboratory has established a series of annual meetings and companion workshops on mouse models for studying human cancer with the overall goal of discussing the development and validation of preclinical mouse models and their use in translational cancer research. This series is designed to support the "Mouse Models of Human Cancer Consortium" (MMHCC) RFA CA-98-013 released by NCI on July 29, 1998. The first meeting, held in October of 1999, focused on mammary carcinogenesis with the specific objective of: 1) evaluating current approaches to the development of mouse models of mammary cancer and 2) their potential application to the development of novel therapeutic approaches to preventing or otherwise treating breast cancer in humans. The second of these meetings will be held at The Jackson Laboratory on October 26-29, 2000 immediately following a companion workshop entitled "Techniques for Modeling Human Cancer in Mice" and will focus on colorectal cancer. Active investigators and students from around the world will have an opportunity to share ideas, establish new collaborations, discuss current progress and chart future directions in the biological analysis of the intestinal epithelium and its neoplasms. Proceedings from this and other meetings in the series will be made available on the World Wide Web.

The objective of this IGERT project is to initiate an interdisciplinary, inter-institutional degree program in Functional Genomics of Model Organisms supported by an interactive faculty from the University of Maine, the Jackson Laboratory, and the Maine Medical Center Research Institute. The major challenge for biological and biomedical research for the foreseeable future is to understand how the information encoded within a genome determines the development and functioning of a living organism. To move from the level of DNA sequence to an understanding of the molecular interplay producing the final traits of an individual will require a continuum of experimental approaches ranging from experimental genomics, molecular biology, and novel biophysical methodologies, to advanced data screening schemes and computational techniques. Traditional alignments of the biologically based disciplines will be insufficient to solve the complex problems associated with functional genomics. Genome projects, regardless of the organism, will rely increasingly on the physical and computational sciences. The increased need for interdisciplinary research will require scientists trained to work interactively in multiple disciplines. This program introduces a new educational paradigm, developed to train students to move freely among the disciplines needed to investigate genome function. Students receive training in the biological, physical and computational sciences through a combination of core and advanced courses, intensive workshops, and research seminars. Emphasis is placed on a high-quality research environment and a tutorial relationship between the student and her/his mentors and program committee. Central to the students' training in interdisciplinary research will be the use of a paired mentoring system, a concept referred to as twinning. The primary mentor plays a role similar to the traditional graduate advisor and comes from the student's primary area of research. The secondary mentor comes from a second discipline, and each student develops a research project dependent upon interdisciplinary collaborations.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the multidisciplinary backgrounds and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In the fifth year of the program, awards are being made to twenty-one institutions for programs that collectively span the areas of science and engineering supported by NSF.

DESCRIPTION (provided by applicant): We are currently in the midst of a genetics revolution that promises to push "breakthrough" biomedical research to new levels. A large part of this revolution depends upon the analysis of the relationships between genes and their effects on complex biological systems and phenotypes. Investigators must become increasingly skilled in the manipulation and use of genetic models to address a variety of important biological questions. The recent decision by President Bush to allow National Institutes of Health funding of human embryonic stem (ES) cell research creates a tremendous opportunity for U.S. investigators. The current major bottleneck to human ES cells becoming a ubiquitous research tool in the biomedical research community is the hands-on training necessary to learn how to culture these sensitive cells. The course proposed in this application, "Current Protocols in Stem Cell Biology", will provide hands-on training for investigators to learn how to culture, manipulate, and differentiate ES cells from human, mouse and zebrafish in vitro. This course will bring together some of the leading experts on ES cell technology and through comparative approaches, effectively train students in the successful culture, maintenance and manipulation of ES cells. A major long term goal for this course will be to share and improve standard protocols, and eventually to publish a technique manual, similar in style to the popular "Manipulating the Mouse Embryo" manual edited by Hogan et al. Specifically, this course will cover: 1) The preparation of media, including necessary quality control; 2) The preparation of feeder layers; 3) ES cell passaging techniques; 4) ES cell cryopreservation; 5) ES cell transfection techniques; 6) FACs analysis of ES cells and their differentiated derivatives; 7) Embryoid body formation; 8) Neural Differentiation of ES cells; 9) Hematopoietic Differentiation of ES cells; and 10) Cardiac Differentiation of ES cells. These aims will be accomplished by offering an intensive 5-day course to 16 participants chosen for their outstanding research potential. The Course will be held on August 5-9, 2002 at The Jackson Laboratory in Bar Harbor, Maine, and will be followed by a companion symposium "Stem Cells on Land and at Sea" to be held at The Mount Desert Island Biological Laboratory on August 9-12.

The Maine EPSCoR Research Infrastructure Improvement award is designed to enhance Maine's competitiveness in molecular biophysical sciences through a partnership between the University of Maine and Maine's non-profit research organizations. The proposed Biophysical Sciences Institute brings together University of Maine faculty in physics, chemistry, biology, mathematics, and spatial engineering, with biomedical researchers at the Jackson Laboratory and Maine Medical Center Research Institute. Maine EPSCoR proposes to hire additional tenure-track faculty in the fields of biophysics and advanced optics, biochemistry, structural biology, applied mathematics, computer science, image analysis and visualization, and material science. The new and existing investigators will form research teams to develop new measurement techniques, new sensors, and innovative approaches to data processing and interpretation in intracellular structures and dynamics, functional materials as a means to manipulate cellular reactions, and biocomputing. In addition to establishing the institute, Maine EPSCoR will integrate research and education through improvements to graduate training.

DESCRIPTION (provided by applicant): We are currently in the midst of a genetics revolution that promises to push "breakthrough" biomedical research to new levels. A large part of this revolution depends upon the analysis of the relationships between genes and their effects on complex biological systems and phenotypes. Investigators must become increasingly skilled in the manipulation and use of genetic models to address a variety of important biological questions. The recent decision by President Bush to allow National Institutes of Health funding of human embryonic stem (ES) cell research creates a tremendous opportunity for U.S. investigators. The current major bottleneck to human ES cells becoming a ubiquitous research tool in the biomedical research community is the hands-on training necessary to learn how to culture these sensitive cells. The course proposed in this application, "Current Protocols in Stem Cell Biology", will provide hands-on training for investigators to learn how to culture, manipulate, and differentiate human ES cells from in vitro. This course will bring together some of the leading experts on ES cell technology to train students in the successful culture, maintenance and manipulation of ES cells. A major long term goal for this course will be to share and improve standard protocols, and eventually to publish a technique manual, similar in style to the popular "Manipulating the Mouse Embryo" manual edited by Hogan et al. Specifically, this course will cover: 1) The preparation of media, including necessary quality control; 2) The preparation of feeder layers; 3) ES cell passaging techniques; 4) ES cell cryopreservation; 5) ES cell transfection techniques; 6) FACs analysis of ES cells and their differentiated derivatives; 7) Embryoid body formation; 8) Neural Differentiation of ES cells; 9) Hematopoietic Differentiation of ES cells; and 10) Cardiac Differentiation of ES cells. These aims will be accomplished by offering an intensive 5-day course to 26 participants chosen for their outstanding research potential. The Course will be held annually in August 3-8, 2003 at The Jackson Laboratory in Bar Harbor, Maine, and will be followed by a companion symposium "Stem Cells on Land and at Sea" to be held at The Mount Desert Island Biological Laboratory on August 9-11.

stem cells; embryonic stem cell; cell bank /registry; biomedical resource; animal colony; laboratory mouse; animal breeding; tissue /cell preparation;

The concept that maternal transcripts govern the physiology of the newly formed embryo is derived from studying non-mammalian organisms with readily accessible oocytes and embryos. The eggs of these organisms usually contain spatially localized proteins and mRNAs, and their embryos become motile and transcriptionally active within hours of fertilization. In contrast, full-grown mammalian oocytes are nonpolar, mammalian embryos do not implant into the uterus until several days after fertilization, and embryonic genome activation occurs a day or more after fertilization. Genomic reprogramming takes place during the oocyte to embryo transition, a process that until now has not been possible to study at a large-scale molecular level in mammals. Our goal is to use the information we derived from the large, representative EST libraries we prepared from mouse oocytes and embryos to explore the mechanisms driving nuclear reprogramming. Timely translation of stored maternal mRNAs provides one mechanism for molecular change in a transcriptionally silent cell. We propose combined computational and molecular approaches to identify cis-elements and their protein partners, which delay translation of maternal mRNAs until oocyte maturation or after fertilization. Retrotransposons may shape and/or inform about the processes that modulate the transcriptional activity of the oocyte and 2-cell embryo. We propose in silico and experimental approaches to explore the effects of retrotransposons on the changing architecture of the embryonic genome.

[unreadable] DESCRIPTION (provided by applicant): Cancer is a genetically complex and biologically heterogeneous group of disorders. It has become increasingly clear that the laboratory mouse, the best genetically defined experimental model organism for humans, presents a major opportunity for rapid advancement in understanding the genetic basis of cancer. Therefore, tools for broad-based training in mouse genetics, genomics and biology is critical to meet this end. The overall goal for our course is to train young scientists (predoctoral, postdoctoral trainees, new investigators) and to re-train established investigators in the use of genetically defined laboratory mice as genetic tools for asking questions about gene function and the role of genetics in cancer. Students completing the course will acquire a working knowledge of: (1) mouse genetics and genomics, (2) growth control and cancer, (3) experimental design and the application of statistical genetics to complex trait analysis, (4) bioinformatics, (5) animal health and ethical considerations in working with mice, (6) basic mouse surgical techniques, (7) in vivo imaging and (8) mouse models for human cancer. How the mouse is used in the translation of basic research to the clinic will be emphasized. [unreadable] [unreadable] These Aims will be accomplished by offering an intensive 10-day course to 30 students chosen for their outstanding research potential. They will interact with a group of prominent mouse geneticists and biologists both from The Jackson Laboratory and other institutions. Student enrollment is kept deliberately small to achieve the desired level of student-faculty interaction. The course will be held annually during the last 2 weeks of August in a retreat setting at Highseas, The Jackson Laboratory's oceanfront conference center that is the site both for the course presentations and residential accommodations for the students and faculty. Lectures, discussions, workshops, and demonstrations are held mornings, afternoons, and evenings for a total of approximately 80 hours of didactic and hands-on training. [unreadable] [unreadable] The Jackson Laboratory is an NCI-designated Cancer Center and has a long history of hosting advanced courses and scientific meetings. The course on "Experimental Genetics of the Laboratory Mouse in Cancer Research" described in this application has been held annually since 1992. [unreadable] [unreadable] Relevance to Human Health: The laboratory mouse is a powerful genetic tool that will continue to play a profound role in understanding the genetic basis of cancer, and in predicting clinical safety and efficacy of new and existing therapies. Short courses such as these are required to develop the cadre of highly skilled young investigators that will be needed to cure this disease. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This is a competing renewal requesting funds for the continuance of the short course "Methods in Human ES Cell Research" formerly known as "Current Protocols in Stem Cell Biology" which has been offered annually at The Jackson Laboratory since 2002. This course will continue to be held in The Jackson Laboratory's Applied Genomics Training Center in early August of each year. This course will continue to bring together some of the leading experts on ES cell technology to train students in the successful culture, maintenance and manipulation of ES cells. Specifically, this course will cover: 1) The preparation of media, including necessary quality control; 2) The preparation of feeder layers; 3) ES cell passaging techniques; 4) ES cell cryopreservation; 5) ES cell transfection techniques; 6) FACs analysis of ES cells and their differentiated derivatives; 7) Embryoid body formation; 8) Neural Differentiation of ES cells; 9) Hematopoietic Differentiation of ES cells; and 10) Cardiac Differentiation of ES cells. The Course will utilize only human ES cell lines registered with the NIH, specifically UC06, WA09 and ES03. One major long term goal for this course will be to share and improve standard protocols, and eventually to publish a techniques manual, similar in style to the popular "Manipulating the Mouse Embryo" manual edited by Hogan et al. In fact, the course syllabus currently contains a compendium of protocols, reagent lists and research papers, providing stem cell researchers with a valuable reference resource. These materials are available upon request from The Jackson Laboratory. These aims will be accomplished by offering an intensive 5-day course to 16 participants chosen for their outstanding research potential. In 2006, the course will be held on August 6-11 at The Jackson Laboratory in Bar Harbor, Maine, and will be immediately preceded by the 2nd Annual International Stem Cell Initiative Workshop at which human ES cell researchers from the major ES cell research centers around the world will meet to discuss the outcome of experiments carried out using shared reagents and methods. The course will be immediately followed by a companion symposium "Stem Cells on Land and at Sea", also held annually, at The Mount Desert Island Biological Laboratory which examines broader issues in comparative stem cell biology. Students in the course are encouraged to attend these companion events free of charge. Relevance to Public Health: The current major bottleneck to human ES cells becoming a ubiquitous research tool in the biomedical research community continues to be the scarcity of the hands-on training necessary to learn how to culture these sensitive cells. The course proposed in this application will continue to provide hands-on training for investigators in how to culture, manipulate, and differentiate human ES cells in vitro. [unreadable] [unreadable] [unreadable]