PROJECT SUMMARY
Rigor and reproducibility in zebrafish research are in jeopardy because current storage methods to
preserve zebrafish germplasm are inadequate. This is especially problematic for the most frequently used
reference strains such as AB, Tübingen (TU), and their hybrid derivatives SAT and NHGRI, which are used for
genome editing applications that critically depend on the accuracy of sequence information. The original DNA
sequences for the four reference strains were generated years ago in select individuals, and random genetic
drift has gradually modified these genome sequences. Other animal models rely on cryopreservation of the
embryo; however, we currently have no working protocols for zebrafish embryo cryopreservation. Despite recent
optimization, sperm cryopreservation – the current method for germplasm conservation – preserves only male-
derived genomes. Although the cryopreserved haploid paternal genome remains stable over time, genome
changes accumulate in live stocks used to provide females. Thus, half the embryo's genome is the same as the
original fish, and half is not.
We will develop an integrated platform for diploid germplasm conservation in zebrafish for the four
reference strains. This platform will help preserve the entire genome close to the published sequence information
or new sequences when they become available. To accomplish this goal, our laboratory at Michigan State
University (MSU) will collaborate with the Aquatic Germplasm and Genetic Resources Center (AGGRC) at
Louisiana State University Agricultural Center (LSUAC) and the Zebrafish International Resource Center (ZIRC)
at the University of Oregon. The new integrated platform for diploid germplasm conservation encompasses two
key steps: 1) isolation, culture, and cryopreservation of diploid somatic cells, and 2) thawing of cells and using
them to derive the original strain by somatic cell nuclear transfer (SCNT – cloning), which is a technique we have
successfully applied to produce founder individuals. This method preserves the sequence of all alleles and
genomes. The specific aims for this project are: Aim 1. Development of an integrated platform to isolate, culture,
cryopreserve and genotype diploid cells from wild-type lines. Aim 2. Standardization of the zebrafish cloning
procedure, including the generation of homozygous zebrafish lines. Aim 3. Promotion, dissemination, and
training of resource center and laboratory personnel using the diploid somatic cell conservation method. At the
end of this R24 proposal, the scientific community will have access to: 1) stable diploid genomes of the most
used reference lines, which is not possible by sperm cryopreservation, increasing the rigor and reproducibility of
experimental results within and between laboratories; and 2) homozygous zebrafish capable of enhancing
studies in cell and organ transplantation, sex determination, and other approaches. We will also show that this
new integrated platform will be capable of: 1) rescuing precious research lines on the brink of extinction when
only adult animals of one sex remain alive, and 2) eliminating pathogens from contaminated lines.