Freezing whole zebrafish embryos in order to later recover live fish is not currently possible. Researchers seeking to preserve zebrafish genetic lines are presently limited to the options of storing frozen sperm in sperm banks or maintaining the fish alive. These limitations increase costs and hinder the efficient dissemination of superior wild type- and mutant strains among investigators. Freeze Back LLC is developing a new approach to the preservation of zebra fish germ-plasm. Somatic cells are collected from embryos at 20 to 22 hours post fertilization and stored at liquid nitrogen temperatures. Live fish are recovered by thawing the somatic cells and using them as nuclear donors for somatic cell nuclear transfer (SCNT). Cloned embryos grow into male and female adults, ready to start producing offspring that carry all the genes of the original line. This technology holds the potential for two important innovations 1) a general method for the routine and effective preservation and recovery of zebra fish germ-plasm and 2) an F1 generation with exactly the same genetic makeup as the original zebrafish line recovered in one step without the need for backcrossing the founder animals. In preliminary studies we have; 1- demonstrated a new and improved method to clone zebrafish from fresh somatic cells isolated from 20 to 22 hpf embryos, 2- cloned healthy fish from cryopreserved cells, and 3- generated animals of both sexes obtained from a single cryopreserved founder. We will offer this new technology as a service. We will routinely store and recover any desired zebrafish strain for researchers at a competitive cost. In Phase II we will demonstrate the generality of these methods by cryopreservation and SCNT cloning of an additional 10 zebra fish strains (including mutant and "weak strains"). Our short-term goal is to develop methods (cryopreservation, thawing and recovery of the original strain by SCNT) that enable the systematic banking of zebra fish. Our long-term goal is to deliver the "Freeze Back system" as a service to the zebrafish research community and as a supplement to currently existing cryobanks e.g ZIRC and EZRC. We will carry out the following aims: Aim I: Clone zebrafish from cryopreserved somatic cells isolated from AB and Tubingen strains. Milestone: Establish multiple first-generation founder animals for these two lines. Aim II: Demonstrate that zebrafish cloned by SCNT from frozen cells, i.e. founder animals, and their offspring are fertile. Milestone: Generate fertile F1 progeny for the two cloned lines. Aim III: Improve the success rate of cloning. Milestone: Achieve a cloning rate of 5%.
Public Health Relevance Statement: Public Health Relevance: Among animal models used for biomedical research, Zebrafish is recognized as the organism of choice by multiple laboratories throughout the world. Approximately 30,000 different zebrafish strains have been created to study development and human disease. Until now, germ-plasm preservation of Zebrafish has been limited to sperm. There is an urgent need to develop the technology that will enable Zebrafish researchers to reliably store and retrieve the strain of choice; Freeze Back LLC has developed a novel system that accomplishes this goal.
Project Terms: Acceleration; Acute; Adult; Aging; Animal Model; Animals; Back; Backcrossings; Biological Preservation; Biomedical Research; Cardiovascular Diseases; Cells; Cloning; Communities; cost; Cryopreservation; Cryopreserved Cell; Development; Embryo; Embryo Cloning; Emerging Technologies; Female; Fertilization; Fishes; Founder Generation; Freezing; Generations; Genes; Genetic; genetic makeup; Germ; Goals; Head; Hematological Disease; Hour; Human; human disease; improved; innovation; Kidney Diseases; Laboratories; Life; Liquid substance; male; Malignant Neoplasms; Methods; Modeling; Mus; mutant; Neurodegenerative Disorders; new technology; NIH Program Announcements; Nitrogen; novel; novel strategies; novel therapeutics; Nuclear; offspring; One-Step dentin bonding system; Organism; Phase; public health relevance; Rattus; Recovery; Research; Research Personnel; response; Services; sex; Somatic Cell; somatic cell nuclear transfer; Source; Sperm Banks; sperm cell; success; System; Technology; Temperature; Testing; Time; Tissues; Uterus; Work; Zebrafish
