Cryopreservation and Cloning of Somatic Cells to Preserve Zebrafish Germplasm

DESCRIPTION provided by applicant 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 to 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 a general method for the routine and effective preservation and recovery of zebra fish germ plasm and an F 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 demonstrated a new and improved method to clone zebrafish from fresh somatic cells isolated from to hpf embryos cloned healthy fish from cryopreserved cells and 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 zebra fish strains including mutant and andquot weak strainsandquot 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 andquot Freeze Back systemandquot 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 F progeny for the two cloned lines Aim III Improve the success rate of cloning Milestone Achieve a cloning rate of

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 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