Advancing nuclear transfer cloning in zebrafish (Danio rerio) into a translational pathway using interdisciplinary tools.

The Zebrafish International Resource Center (ZIRC) is an NIH-funded national stock center and germplasm repository that maintains and distributes genetically modified and wild-type zebrafish (Danio rerio) lines to the biomedical research community. The ZIRC and its community would benefit from incorporating somatic cell nuclear transfer (SCNT) cloning which would allow the preservation of diploid genomes. The goal of this study was to advance a zebrafish SCNT cloning protocol into a reproducible community-level pathway by use of process mapping and simulation modeling approaches to address training requirements, process constraints, and quality management gaps. Training, for most steps in the SCNT protocol, could be completed within two months; however, steps that involved micromanipulation of eggs required more than four months of training. Dechorionation of embryos and egg micromanipulation were identified as major constraints because the processes were performed manually and required advanced operator manual skills. Chemical dechorionation and microfluidic devices to aid micromanipulation were identified as ways to eliminate these constraints. Finally, quality control steps to record the initial quality of collected germplasm were recommended to prevent production defects and harmonize the SCNT pathway across multiple facilities. By beginning to enhance the reproducibility of the SCNT cloning pathway, this technique can be implemented across zebrafish research facilities and facilities that work with other biomedical models.