Zoltan M. Varga - US grants

DESCRIPTION (provided by applicant): In the past decade, laboratories around the world have produced tens of thousands of mutant, transgenic, and wild-type fish lines. Maintaining these valuable genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. One of the major recommendations of the 2007 NCRR workshop 'Achieving High-Throughput Repositories for Biomedical Germplasm Preservation' was for performance of detailed studies to improve sperm cryopreservation for important aquarium fish models. Pursuant to this we propose to establish high-throughput processing of zebrafish sperm for stock centers and laboratories by extending a comprehensive cryopreservation pathway, and by intensifying efficiency and output to enable implementation of germplasm repositories. The Specific Aims are to: 1) broaden the pathway to improve quality and provide multiple access points for samples produced by current approaches, 2) intensify the pathway for automated processing and repository development including use of industrial modeling and decision-making tools, and 3) implement two scalable cryopreservation protocols; the large-scale high throughput method for Resource Center use and, reverse-engineered from it, a small to intermediate scale application for other research laboratories to back up their own lines or submit them to the Resource Center. This will improve quality, reduce variability, and increase efficiency. We propose to improve broodstock conditioning, gamete quality, fertilization and reproduction, and to accommodate the variety of approaches, levels of application, and current and future needs of the research community. This will yield two complementary approaches: one at an industrial scale using automated equipment and pooling of samples to yield the highest level of intensity and efficiency; the other, less intensive but highly reliable, designed or dissemination to other laboratories. The materials generated from this external pathway will feed into the central pathway and will provide an additional form of throughput based on the aggregate production from multiple locations. A high level of standardization will be necessary for both approaches to merge efficiently to deliver high-quality samples into large comprehensive germplasm repositories. Thus, we envision this renewal as a funnel to improve and adapt the wide breadth of technical approaches available for zebrafish cryopreservation, and to focus these activities for repository development that can be eventually coupled with commercially available high-throughput equipment, supplies, and reagents specifically optimized for aquarium fish. This approach is practical and extremely feasible. This renewal proposal builds directly on the success of our established pathway and looks to couple efficiently with future advances. PUBLIC HEALTH RELEVANCE: Aquarium fish models are rapidly transforming genetic research of disease. Thousands of new research models become available each year, and we must develop the abilities and resources necessary to preserve them in repositories for biomedical research and improvement of human health. The most significant problems constraining repository development for these fish are the lack of automated, high-throughput cryopreservation capabilities, and the lack of standardized protocols and terminology. These deficits prevent the coupling and integration of activities among different laboratories and stock centers.

A & R Supplement Request for R24?OD010441 Summary Our current R24 is intended to establish reproducibility of cryopreservation results for the aquatic biomedical research community through development of novel devices that address the major problems in achieving standardization. This will improve efficiency, output, and quality. These devices are being engineered through microfabrication and 3?D printing to ensure that they are inexpensive, effective, and available through open?source file sharing. The goal is to provide a comprehensive standardized approach for cryopreservation that can enable routine participation by all members of the zebrafish research community in a form of aggregate throughput that can accommodate existing protocols, integrate novel approaches, and enable development of germplasm repository networks. This technology?development process is intended to also be suitable for other aquatic biomedical models, and compatible for use with traditional mammalian models such as mice. We have made good progress during year 1 (e.g., >10 papers published or in press) in design and prototyping of novel devices. This is grounded in our establishment of a rich interdisciplinary culture of engineering innovation directed at biological problems associated with repository development. Based on this success we have identified specific needs and bottlenecks that can be addressed to expand the throughput and scale of the current R24 project. We propose to create an Advanced Fabrication Laboratory by repurposing of a concrete structure (formerly a bull barn) currently used as a storage space (2,660 ft2) attached to our main building at the AGGRC (renovation budgeted at $123,000, see Budget Justification). We also propose to purchase large fabrication equipment ($25,000; items >$5,000) and small equipment ($25,000; $1,000?$5,000) to increase our scale and expand our capabilities. In short, this would improve our current R24 project by allowing us to: 1) efficiently increase production of effective device designs for advanced testing; 2) expand fabrication capability to produce custom parts with technologies in addition to 3?D printing (e.g., milling, thermoforming, laser engraving) and with additional materials (e.g., metal) to produce composite devices; 3) incorporate smart technology into the devices (e.g., Bluetooth, WiFi, motor control), and 4) incorporate smart processing (e.g., web cameras, design file sharing) into our fabrication processes to gain greater interaction with user communities. All of these activities are contained within the scope of the current R24 project, yet would greatly increase our productivity and scale. They would also directly enable production of sample products that could be beta tested at a large scale by user groups through other funding.