Bodenstein Sarah, Poulos William, Jimenez Fermin, Stout Michael, Liu Yue, Varga Zoltan M, Cibelli Jose, Tiersch Terrence R
Louisiana Sea Grant College Program, Louisiana State University, Baton Rouge, LA, United States of America.
Aquatic Germplasm and Genetic Resources Center, School of Renewable Natural Resources, Louisiana State University Agricultural Center, Baton Rouge, LA, United States of America.
PLoS One. 2024 Dec 30;19(12):e0312672. doi: 10.1371/journal.pone.0312672. eCollection 2024.
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.
斑马鱼国际资源中心（ZIRC）是一个由美国国立卫生研究院资助的国家种鱼中心和种质库，负责维护并向生物医学研究界分发转基因和野生型斑马鱼（Danio rerio）品系。ZIRC及其社群将受益于纳入体细胞核移植（SCNT）克隆技术，这将有助于保存二倍体基因组。本研究的目标是通过使用流程映射和模拟建模方法来解决培训要求、流程限制和质量管理差距，将斑马鱼SCNT克隆方案推进到可重复的社群级流程。对于SCNT方案中的大多数步骤，培训可在两个月内完成；然而，涉及卵子显微操作的步骤需要四个多月的培训。胚胎去绒毛膜化和卵子显微操作被确定为主要限制因素，因为这些过程是手动进行的，需要操作人员具备先进的手工技能。化学去绒毛膜化和辅助显微操作的微流控设备被确定为消除这些限制的方法。最后，建议采取质量控制步骤来记录所收集种质的初始质量，以防止生产缺陷并使多个设施的SCNT流程保持一致。通过开始提高SCNT克隆流程的可重复性，这项技术可以在斑马鱼研究设施以及使用其他生物医学模型的设施中实施。
