Recipient Organization
MOTE MARINE LABORATORY
1600 KEN THOMPSON PARKWAY
SARASOTA,FL 34236
Performing Department
(N/A)
Non Technical Summary
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.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Goals / Objectives
Our overall goal is to develop embryonic cell lines from red drum (Sciaenops ocellatus) and Pacific whiteleg shrimp (Litopenaeus vannamei).Objective 1. Collection of fertilized embryos from red drum and Pacific whiteleg shrimp. Red drum broodstock will be spawned and embryos collected at various stages post-fertilization, with an emphasis on collecting embryos at the blastomere stage.Objective 2. Embryonic stem cell isolation. Embryonic stem cells will be isolated from fertilized eggs of each of the proposed species.Objective 3. Thraustochytrid control. Control of thraustochytrid spp. growth in marine invertebrate cultures is necessary; this objective will test a panel of antibiotics/antimycotics that are effective against thraustochytrid but do not have a negative impact on target cell growth.Objective 4. Biochemical characterization of egg contents. The biochemical composition of fertilized eggs will be analyzed with regard to fatty acid and amino acid profiles, total carbohydrate, as well as proximal analysis.Objective 5. Transcriptomic analyses. In this objective, we will conduct transcriptomic experiments to characterize cellular and genomic events occurring in cells at different stages in culture and in response to different media formulations.Objective 6. Spent media analysis. Nutrient utilization in cell culture samples will be analyzed at the same time points and media conditions as for transcriptomic analysis.Objective 7. Species targeted growth factors. In this objective, we will characterize growth factors based on sequence homology to identify homologous growth factors in the genome of each species.Objective 8. Morphological monitoring of cell growth over time. Cell growth properties, proliferation, and cell death will be monitored over time in cells cultured in different media formulations.Objective 9. Optimize media formulations. In this objective, we will develop medium formulations based on nutrient composition (Objective 4), growth factor analysis (Objective 7), spent media analysis (Objective 6), and results from monitoring cell growth over time (Objective 8).
Project Methods
Our overall approach to developing cell lines is based on collection of fertilized eggs at the blastomere stage, with isolation of embryonic stem cells. Fertilized red drum eggs will be collected from broodstock reared at Mote Marine Laboratory. Fertilized shrimp eggs at the blastomere stage will be acquired through collaboration with Florida-based commercial shrimp farmers. Our approach to isolating stem cells from fertilized eggs to initiate cell lines is based on generally recognized important features of embryonic stem cells, such as high proliferation and pluripotency that are amenable to cell line development. The approach is targeted and species-specific. Efforts to facilitate this approach are first to understand nutrient requirements for cells of each species by characterizing egg nutrient composition that supports cell growth initially in the fertilized eggs and analyzing media components in cultured cells (spent media analysis) to understand the primary nutrients consumed by cultured cells. Furthermore, a transcriptomics approach will provide valuable genomic data for uncharacterized species and identify important changes in gene expression that determine whether a cell dies or progresses towards an immortalized cell line. Development of a cell culture media that drives expression of those genes will help us meet our end goal. This project will also ensure target cell types through genetic markers and control of contaminants specific for invertebrate cell culture.The success of our project will be determined by tangible results that are obtained. The ultimate tangible result is a continuously growing cell line from each species, that can be cryopreserved, shared, and recultured in other laboratories. Other measures of success include successful inhibition of thraustochytrid growth, identification of species-specific growth factors that improve cell growth, and identification of important genetic events that lead to greater duration in cell cultures.Knowledge resulting from these research efforts includes the additional tools that will be available for researchers in the field to use as a framework and a guide for generating additional cell lines from other seafood species of commercial relevance.