Generation and validation of antibodies and antibody-based biosensors for the zebrafish research community

Project Summary Zebrafish are a powerful model system for biomedical research that offers many advantages over other animal models. The tiny freshwater fish are relatively inexpensive to maintain, easy to breed in large numbers, and their embryos are transparent, which makes them highly amenable to non-invasive microscopic imaging at single cell resolution. Furthermore, the development of approaches to genetically manipulate zebrafish has rapidly accelerated this model system’s utility and contribution to research breakthroughs, including those uncovering molecular mechanisms driving human diseases. Despite these numerous advantages, research progress using zebrafish as a model system has suffered from the lack of available, reliable, reproducible, molecularly defined antibodies targeted to zebrafish proteins. As a result, mechanistic cell biological and biochemical studies are often incomplete or missing from zebrafish studies leading to incomplete mechanistic characterizations of proteins and processes and overall reduced reproducibility and rigor in zebrafish research. To address these problems, we have developed low-cost, accessible tools and methodologies to design and produce recombinant monoclonal antibodies from primary sequences using high growth density suspension culture cells. In addition, we developed strategies to diversify recombinant antibodies to generate new products including species specificity variants and genetically encoded scFvs (single chain variable fragments, or nanobodies), which can be used as biosensors in living cells. In Aim 1, we will develop, characterize, and validate recombinant antibodies against zebrafish proteins whose sequences are invariant and molecularly defined. We will also generate species variants of each antibody to increase experimental flexibility. In Aim 2, we will develop scFv-based biosensors for select zebrafish target protein post-translational modifications. These biosensors, which will be genetically encoded and fluorescently-tagged, are ideal tools for real-time tracking of post-translationally modified epitopes in living cells and whole organisms. In Aim 3, we will build, maintain, and promote an online resource to provide information, protocols, and reagents produced from this resource grant. This will serve as the portal to supply antibodies at cost to the zebrafish community and to provide protocols and reagents to labs that wish to produce their own recombinant antibodies. By combining complementary expertise from two laboratories (PI Ramani Ramchandran, PhD – zebrafish model expert, Medical College of Wisconsin; and PI Jennifer DeLuca, PhD – antibody engineering expert, Colorado State University), we aim to generate a publicly-available library of high-quality, validated, cost-effective, and immortal source of antibodies to the zebrafish community, which will significantly increase rigor, reproducibility, sustainability, and accessibility in zebrafish research.