Robert E. Braun - US grants

DESCRIPTION (provided by applicant): The production of gametes in mammals is an elaborate process that begins during embryogenesis and continues during the reproductive life of the organism. Gametogenesis involves the production of highly specialized cells with unique organelles designed to accomplish the union of sperm and egg at fertilization. The timely production of developmental^ important products involves the regulated translation of mRNAs that have accumulated earlier during gametogenesis. Deviation from the wild-type timing of translation can cause cessation of gametogenesis and lead to sterility. The goals of this proposal are to further our understanding of the mechanisms of translational repression and activation during mammalian spermatogenesis. The studies in this proposal will focus on the regulation of posttranscriptional control in adult male germ line cells in mice. In Aim 1, the function of the Y box proteins MSY2 and MSY4 will be investigated using conditional gene targeting. In Aim 2, MSY2 and MSY4-interacting proteins will be identified by mass spectrometry analysis of ribonucleoprotein particles precipitated with MSY2 and MSY4 antibodies. Prm1-enriched mRNPs will be isolated using biotinylated antisense RNAs and streptavidin-coated immunomagnetic beads. In Aim 3, proteins identified by mass spectrometry will be verified using complementary methods and functional studies will be pursued in cell culture and in vitro to determine how the associated proteins contribute to translational repression and mRNA stability. The knowledge gained from these studies may be useful in the genetic assessment of male infertility in humans, and lead to the development of male contraceptives designed to disrupt essential regulatory steps during normal spermatogenesis.

A Research Experiences for Undergraduates (REU) Sites award has been made to The Jackson Laboratory (JAX) that will provide a residential summer research experience for 10 undergraduates and 2 incoming college freshmen per year. The program focus is functional genomics, a rapidly evolving scientific discipline that seeks to understand how the information encoded within a genome determines the development and function of a living organism. Under the guidance of 11 available faculty mentors, the program challenges students with hands-on projects that utilize diverse interdisciplinary approaches linking the biological, physical, and computational sciences to investigations of genome function. A strong emphasis is placed on ethics education and professional development training, and institutional commitment includes student access to on-campus courses and conferences, state-of-the-art instrumentation, dedicated program direction by the Education Office, and housing in a JAX-owned facility overseen by professional staff. The REU program is as an integral part of the 10-week JAX Summer Student Program (SSP), an internationally recognized research opportunity that annually welcomes 30-40 undergraduates and high school students. A weekly stipend, subsistence allotment, and travel assistance provided to each intern enables economically disadvantaged students to forgo summer jobs and participate in this life-changing educational experience. The SSP has well-established administrative procedures for recruitment, program design and management, mentor guidance, and alumni tracking to determine lasting influences of the research experience. REU participants are chosen through a competitive, nationwide application process that targets students with a strong interest in functional genomics. Members of underrepresented groups are encouraged to apply and actively recruited, and at least half are selected from academic institutions where research opportunities in STEM are limited. Professional third-party evaluation is conducted by the University of Maine, and the program is guided by the common assessment tool available to BIO-funded REU PIs. More information is available at http://education.jax.org/summerstudent/index.html, or by contacting PI Robert Braun, Ph.D., or Lisa John, Training Coordinator, at (207) 288-6924 or summerstudents@jax.org.

DESCRIPTION (provided by applicant): The Bioinformatics/Computational Biology (BI/CB) investigators at The Jackson Laboratory are national leaders in community database development, statistical genetics, and the development of computational approaches for understanding gene function. Biomedical researchers here and in the wider scientific community are tapping the wealth of mouse sequence and phenotype data to understand the molecular networks involved in human disease and normal development. In response to the increased reliance of functional analyses on computational approaches, the BI/CB programs are expanding to add strength in computational biology and to further integrate in silica and in vivo approaches for understanding gene function. In the long term, such collaborative, interdisciplinary research will be enabled by the development of nationwide computing grids. Richly annotated data from our substantive phenotyping and genetic characterization programs, accompanied by tools for data visualization and mining, will add to the national endeavor. To achieve that goal, Jackson plans to recruit four new faculty members over the next five years with expertise in applying computational approaches to solving biological problems. However, our three-floor BI/CB area is at capacity because of the rapid growth of existing programs. There is no space for new recruits or expected program expansions. In addition, there is insufficient space to house the expanding computing hardware necessary to meet the increased needs of the BI/CB programs and our wet-bench research programs, which are increasingly dependent on computation. The project described in this application will create the additional office and conference space necessary to support expansion of the BI/CB programs. It will also construct a Computing Infrastructure Facility creating the additional space necessary to house the existing and anticipated additional computing hardware required to support the increasingly data- and computationally intense research of the entire Jackson Laboratory scientific staff.

? DESCRIPTION (provided by applicant): The Jackson Laboratory (JAX) has been a leader in mammalian genetics research and education since its inception in 1929. JAX pioneered the development of genetic approaches toward understanding fundamental questions in mammalian development and the genetic underpinnings of human disease. JAX also has a long and illustrious track record of training postdoctoral-and more recently predoctoral-scholars to be productive researchers at the nexus of systems genetics, genomics and computational biology. To support JAX's dual mission of discovery and education, we continually strive to provide cutting-edge, unique and highly relevant training opportunities designed to prepare all JAX trainees for successful scientific careers. The NICHD T32-funded Training Program in Developmental Genetics (TPDG), funded since 1977, is integral to the postdoctoral training environment at JAX. This developmental genetics-focused program provides high-quality postdoctoral training to prepare its trainees for careers as independent investigators in universities, research institutions and the biomedical industry. Today, the program brings together 18 highly productive and well-funded faculty mentors at the JAX Mammalian Genetics campus in Bar Harbor, ME, who provide a uniquely interdisciplinary, integrated and comprehensive training environment. Research areas include mammalian embryonic development, congenital birth defects associated with neurological, sensory and metabolic disease, stem cell biology, reproduction, systems genetics and bioinformatics. The TPDG has supported ~70 postdoctoral trainees to date, 20 during the past 10 years. Previous trainees have been highly successful in moving on to careers in academia, government and industry, and in obtaining research funding. In this resubmission of the TPDG we seek to continue to provide two slots for postdoctoral fellows and request support for two predoctoral students. JAX participates in collaborative Ph.D. training programs with the Tufts School of Medicine and the University of Maine that have grown to critical mass. Specific criteria are used to admit both pre- and postdoctoral trainees into the TPDG. Required and elective curricula have been developed to enrich the training experience. Trainees will test hypotheses regarding the genetic underpinnings of development and disease, and will model new diagnostic and therapeutic modalities, using cutting-edge computational and mouse genetics resources uniquely available to them at JAX. Trainees will have access to focused and individualized career development opportunities, such as teaching and grant writing experiences, and will be integrated into seminars, workshops, and research interest groups. They will write external funding applications, present their findings at scientific meetings, and publish in peer-reviewed journals. Trainee evaluation will be rigorous and coupled with support for growth. The TPDG will thus provide exceptional research opportunities in a stimulating scientific training environment and enable trainees to launch successful independent careers in biomedical research.

DESCRIPTION (provided by applicant): The Jackson Laboratory (TJL) Cancer Center has 58 members organized into one Research Program, Modeling Cancer: Stem Cells to Therapy. Research is focused on understanding the fundamental mechanisms of cancer initiation and progression in the context of mouse biology and genomics. The mouse is the best experimental mammalian system for examining cancer both at the level of individual genes that participate in cancer initiation and at the genome-wide scale that influences individual susceptibility. Research performed at TJL Cancer Center draws on the unparalleled mouse resources supported by the Center, and in turn enhances both the genetic and information resources maintained at TJL for use by TJL Cancer Center members and the wider cancer research community. The cutting edge instrumentation and technically sophisticated Scientific Services supported by the Cancer Center enable all members to conduct experiments that may require expertise and equipment that is not available in their own laboratories. The scientific leadership of TJL has direct oversight of the Cancer Center. Dr. Richard Woychik is Director of TJL and the Cancer Center. Dr. Barbara Knowles is Vice President for Education and Collaborations of TJL and Deputy Director and Program Leader of the Cancer Center. They receive external advice from the Board of Scientific Overseers for TJL and the Cancer Center. Several internal advisory committees composed of Cancer Center members and senior managers ensure that the Cancer Center is a priority in budgeting, recruiting, and overall operations at TJL. Support is requested for Scientific Resources and Services: a) the Mouse Models Resource,, which provides genetically defined mice to Cancer Center Members;b) Computational Sciences, for expertise and analytical tools for advanced computational and statistical analysis;c) Genome Sciences, for tools for genome scanning, allele typing, and sequencing;d) Histopathology and Microscopy Sciences, for electron and light microscopy, cytogenetics, necropsy and histology;e) Phenotyping Sciences for gene expression, molecular biology, flow cytometry, and protein chemistry;f) Reproductive Sciences for cell biology and microinjection services, and g) Reproductive Sciences for rederivation, cryopreservation and reconstitution of mice. Funds are also requested for planning, emphasizing translational and transdisciplinary research. Developmental funds are requested for a pilot project program to stimulate new cancer research opportunities and for new investigators who will bring additional strength to TJL Cancer Center's focus on cancer in the context of mouse biology and genomics.

DESCRIPTION (provided by applicant): The Research Animal Facility (RAF) is central to the research conducted at The Jackson Laboratory (JAX). JAX principal investigators currently hold 138 awards totaling over $47.9 million annually for research using mice. The oldest portion of the RAF dates from 1947, and has been modified ever since as research needs changed, mouse husbandry practices evolved and the importance of defined health status was recognized. In the past 10 years, JAX has made nearly $6 million in improvements to the RAF to accommodate these changes. Emerging trends in mammalian genetics/genomics continue to drive change in patterns of mouse use at JAX. First, new mouse models for modeling human genomic variation have been developed at JAX, and are maintained in core resource colonies supported by large center grants. Researchers use these colonies to identify loci participating in a variety of complex traits, and then maintain smaller, individual colonies to refine map positions, identify and validate candidate genes. Second, many JAX investigators are using smaller colonies of specialty mutant strains of mice to conduct functional analyses on a variety of biological and molecular processes. Third, functional assessments and potential therapeutic interventions for human disease must be verified in human tissues. Advances in immunocompromised mouse hosts pioneered at JAX enable long-term engraftment of a variety of human tissues. All of these research applications require 1) high barrier animal holding rooms where the health status of experimental mice can be consistently maintained, and 2) flexible procedure space where phenotypes can be assessed, experimental manipulations performed, and tissues collected. A cross-institutional planning committee has developed a long-range plan for the RAF to meet these and other anticipated needs. This proposal requests partial funding for the first step in this plan. JAX will commit institutional funds, expected to total over $950,000, to complete the project. As the average size of individual mouse colonies maintained in the RAF decreases, JAX can reconfigure space to provide more flexible procedure space and modify existing animal holding rooms to allow operation as high barriers. To meet this objective, we propose to: 1) upgrade rooms 2845 and 2835 in Building 20 to high barriers, by purchasing and outfitting them with positively individually ventilated (PIV) caging system and ventilated changing stations. A dedicated clean corridor accessed via an existing air shower-equipped personnel entry/exit lock will service these rooms. 2) expand procedure room space by creating and equipping three procedure rooms totaling 455 ft2. The rooms are designed to offer maximum flexibility of use to accommodate increasingly complex and specialized research procedures. Two rooms will be equipped with Baker NCB-D4 Vertical Flow Biological Safety Cabinets. One will be furnished with mobile workstations to maximize flexible configuration. The project will be overseen by John Fitzpatrick, P.E., Senior Director of Facilities Services, who will serve as Facilities Person/Project Manager.

DESCRIPTION (provided by applicant): The Jackson Laboratory (JAX) is well positioned to fully participate as a member of the KOMP2 Research Network and support its objectives to build a global resource for understanding mammalian gene function. JAX is a leading center for mouse genetics and the development of mouse models of human disease. Drawing from deep cumulative knowledge and the unique spectrum of expertise of Jackson Laboratory scientists, we have assembled the necessary leadership and created a blueprint for a cost-effective JAX KOMP2 Phenotyping Center that will produce high quality functional assessments of KOMP strains. Demonstrated experience and proven success in numerous previously funded high throughput phenotyping programs at JAX adds value and efficiency to our proposed Center. JAX also offers an unparalleled track record for animal production, husbandry, evaluation and data management required to meet the challenges of this project. The JAX KOMP2 Phenotyping Center will: 1) obtain primary, per-mouse data for 1,250 mutant strains across 235 phenotypes per strain; 2) provide quantitative and qualitative assessment of phenotypes for each mutant strain, using automated calling procedures, statistical, technical and scientific review; 3) deliver phenotypic data to the KOMP2 Data Collection Center in a timely and efficient manner; and 4) develop innovative approaches to increase the efficiency of phenotyping processes and the value of these data to the scientific community. We present a dual-pipeline strategy to collect essential physiological data from 2 cohorts of each KO strain, followed by in-depth necropsy and histology to complete the assessment of multiple organ systems. One pipeline is devoted to evaluation of behavior, neurophysiology, vision, sleep and hearing, and a second pipeline captures metabolic, cardiovascular and immune function. We have augmented the value of the pipeline design by adding phenotypes underrepresented in large-scale efforts, such as sleep, and by integrating opportunities for innovation and technology development to further streamline the process and identify ways to reduce overall costs. We have assembled a panel of Domain Experts to provide routine quality assurance of all data and participate in comprehensive assessments for each KO strain. This effort will promote the identification of KO strains as models for human disease and provide insight into functional consequences of eliminating a single gene.

PROJECT SUMMARY In response to ?NOT-RM-20-015?, we propose an urgent competitive revision to The Jackson Laboratory Knockout Mouse Phenotyping Project (KOMP2) to develop the next generation of precision mouse models for mechanistic discovery of SARS-CoV2 infection and therapeutic discovery of COVID-19 disease treatments. The worldwide response to the COVID-19 pandemic has triggered a surge in demand for animal models to understand the underlying biology and pathology of SARS-CoV-2 infection and for the preclinical development of novel therapeutic strategies. Several mouse models have recently been reported that respond with varying degrees to SARS-CoV-2 infection. However, given the diversity of patient outcomes, any one mouse model on a standard inbred genetic background does not reflect the impact of host genetic context on SARS-CoV-2 infection and response to treatment. We therefore propose to create a second-generation mouse model platform incorporating diverse genetic backgrounds, to characterize the variation in SARS-CoV-2 infection dynamics and the development of clinically-relevant disease. The proposed project will rapidly provide the research community with an urgently needed resource for linking the variability in COVID-19 disease outcome with underlying host genetic features, and for developing precision therapies tailored to treat the individual patient. Our Specific Aims are: 1) To create a panel of genetically diverse transgenic models for SARS-CoV-2 infection; 2) To characterize infectivity, phenotypic response, disease outcome, and transcriptome heterogeneity of these models; and 3) To distribute these models and our outcome data to the scientific community.

PROJECT SUMMARY/ABSTRACT Most adult tissues are maintained by resident adult stem cells that maintain the function and integrity of the tissue. As old cells die or are damaged, new cells are produced from adult stem cells. In many tissues there is emerging data suggesting the presence of both rapid-cycling and quiescent stem cells. Rapid cycling cells are actively engaged in tissue repair while slow-cycling, or G0-arrested cells, are reserve cells. Recently published work, and preliminary studies in this proposal, support the hypothesis that spermatogonial stem cells (SSCs) in the testis are heterogeneous in their cycling status and that disruption of the normal cell cycle can interfere with both self-renewal and differentiation. We have identified a subpopulation of spermatogonia that express EOMES, a T box transcription factor. Using lineage tracing we have shown that they contribute to steady-state spermatogenesis and to regeneration following germ cell ablation by busulfan. In Plzf mutant mice, which show an age-dependent depletion of SSCs, EOMES+ cells cycle more rapidly, suggesting that age-dependent depletion of SSCs is caused by proliferative exhaustion. The central hypothesis of this proposal is that spermatogonial stem cells (SSCs) are also heterogeneous with respect to their cycling status and that there are both rapid cycling and slow cycling SSCs. We propose that proper regulation of the cell cycle is critical for maintaining the homeostatic balance between self-renewal and differentiation and that loss of cell cycle regulation can lead to age-dependent loss of SSCs due to the mis-regulation of critical self-renewal genes. In Specific Aim 1 we will quantify the frequency of cell cycle asynchrony, the concordance of SSC marker expression and cycling status, and the effect of mutation of Plzf on both of these parameters. In Specific Aim 2 we will utilize an allele of Ki67 (Mki67-RFP) to flow sort cycling and non-cycling cells from the larger pool of GFRA1+ population. Single cell RNA sequencing (scRNAseq), and single nuclei Assay for Transposase Accessible Chromatin (snATACseq) of the cycling and non-cycling cells will provide and independent assessment of the non-cycling G0 population of SSCs, whether Eomes and other markers of SSCs cells are enriched within the population, and whether there is a previously unidentified population of G0 cells within the SSC pool. Lastly, In Specific Aim 3 we will assess how mutation of Batf, a regulator of Eomes expression, enhances the germ cell loss phenotype in Plzf lu/lu mutants, and assess the role of the mis-regulation of cell cycle genes in the driving the hyperproliferative phenotype.