As the zebrafish research community grows, an increasing number of shared resources are generated. At the ZIRC, adjustments are continually made to accommodate these changes and maintain a balance between efficient resource regeneration and effective distribution. For example, the inventory of mutant and transgenic lines at the ZIRC has increased by over 1000% within the last five years. This increase impacted greatly the strategies undertaken for line identification, regeneration, quality control, and distribution. Previously, most fish lines were mono-allelic. A large number of these lines were maintained as stocks consisting of identified transgene and mutation carriers. The lines were distributed as genotyped adult fish or embryos obtained from natural crosses. Now, most of the ZIRC lines are multi-allelic, and they arrive as frozen sperm. Distribution of these lines involves shipment of embryos generated by in vitro fertilization (IVF) using thawed sperm. We rederive lines to amplify samples in the ZIRC sperm bank. This
process depends strictly on the demand. Lines frequently requested are regenerated more often than lines that are seldom ordered. Regeneration of multi-allelic mutant lines required a significant increase of the genotyping throughput at the ZIRC. We handled this in part by using Kompetitive Allele Specific PCR (KASP) genotyping (LGC Genomics). With this technique, genotypes are determined based on a fluorescence detection system rather than time-consuming restriction enzyme digestion and gel electrophoresis. Due to the immense increase in the number of imported lines, we perform quality control for new imports primarily during line regeneration rather than at the time of importation as previously. Thus, the ZIRC staff no longer confirms all of the distributed genotypes. To reflect the status of ZIRC sperm samples with regard to line identification, we now provide sperm sample
categories. These include a category for samples derived from males whose genotypes were confirmed at the ZIRC as well as a category for samples with genotypes confirmed elsewhere. Only lines with genotypes verified at the ZIRC are distributed without a disclaimer. Lines genotyped elsewhere or whose genotypes have not been confirmed are offered with specific disclaimers.