** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** DEVELOPMENT OF CELL LINES IS CRITICAL IN MANY RESEARCH FIELDS SUCH AS DISEASE, REPRODUCTION, GENETICS, AND BIOTECHNOLOGY. CELL LINES ARE VERY USEFUL IN VITRO MODELS FOR TOXICOLOGICAL, PATHOLOGICAL, AND IMMUNOLOGICAL STUDIES. THE RESULTS FROM THE PROPOSED STUDIES WILL INCLUDE ESSENTIAL TOOLS FOR RESEARCHERS TO USE AS A FRAMEWORK AND GUIDE FOR IMPROVING CELL LINE DEVELOPMENT IN AQUATIC MARINE SPECIES. THE RESEARCH WE PROPOSE WILL LEAD TO THE DEVELOPMENT OF CELL LINES FROM ONE FISH (RED DRUM SCIAENOPS OCELLATUS) AND ONE SHRIMP (PACIFIC WHITE SHRIMP, LITOPENAEUS VANNAMEI) SPECIS. OUR APPROACH IS BASED ON ISOLATING EMBRYONIC STEM CELLS (ESCS) FROM FERTILIZED EMBRYOS AT THE BLASTOMERE STAGE. WE HYPOTHESIZE THAT IMPORTANT FEATURES OF ESCS, SUCH AS HIGH PROLIFERATION AND PLURIPOTENCY, WILL FACILITATE DEVELOPMENT OF A CONTINUOUS CELL LINE THAT CAN ULTIMATELY BE USED IN A VARIETY OF RESEARCH APPLICATIONS. WE ALSO PROPOSE A WHOLISTIC AND SPECIES-SPECIFIC TARGETED APPROACH, BASED ON NUTRIENT NEEDS OF ISOLATED EMBRYONIC CELLS (NUTRIENT ANALYSIS OF EGGS) AND CELLS IN CULTURE (SPENT MEDIA ANALYSIS), USING A TARGETED APPROACH TO GROWTH FACTOR IDENTIFICATION WITH A HIGH PROBABILITY OF SUCCESS BASED ON CONSENSUS SEQUENCES THROUGH BLAST, UNDERSTANDING EVENTS AT THE CELLULAR AND GENOMIC LEVEL (TRANSCRIPTOMIC ANALYSIS TO DETERMINE THE TIMELINE OF ACTIVATION FOR PATHWAYS OF CELL PROLIFERATION AND/OR CELL DEATH). THE FRAMEWORK USED TO DEVELOP THESE CELL LINES WILL ALSO BE APPLICABLE TO OTHER SPECIES OF INTEREST, INCREASING THE NUMBER OF CELL LINES AVAILABLE AND NEEDED FOR VARIOUS INTERDISCIPLINARY RESEARCH INTERESTS.A SIGNIFICANT CONTRIBUTION OF THIS PROJECT WILL BE TO INCREASE THE COMPETITIVENESS OF THE US IN THE FIELD OF AQUACULTURE BY MAKING CELL LINES AVAILABLE FOR RESEARCH IN KEY AREAS OF LIKE HEALTH, DISEASE DIAGNOSIS, SAFETY, AND NUTRITIONAL ASPECTS CHALLENGING AQUACULTURE PRODUCTION. INCREASING THE NUMBER OF CELL LINES RESEARCH CAN BE CONDUCTED USING THESE CELL LINES WITHOUT SCARIFYING WHOLE LIVE ANIMALS. THE SCIENTIFIC KNOWLEDGE GENERATED USING FISH CELL LINES WOULD BE IMMENSELY USEFUL FOR QUALITY PRODUCTION IN A SUSTAINABLE MANNER. CELL LINES WOULD FACILITATE IN VITRO RESEARCH FOR DEVELOPING CLIMATE-RESILIENT AND SUSTAINABLE AQUACULTURE SYSTEMS TO MINIMIZE THE KEY CHALLENGES AND PROVIDE NUTRITIONAL SECURITY TO THE BURGEONING WORLD POPULATION.