Establishing an Integrated Platform for Diploid Germplasm Conservation in Zebrafish (Danio rerio)

项目摘要 斑马鱼研究的严谨性和可重复性处于危险之中，因为目前的储存方法， 保存的斑马鱼种质是不够的。这对于最常用的 参考菌株，如AB、Tübingen（TU）及其杂交衍生物SAT和NHGRI，用于 关键依赖于序列信息准确性的基因组编辑应用。原始DNA 四种参考菌株的序列是多年前在选定的个体中产生的， 漂变逐渐改变了这些基因组序列。其他动物模型依赖于冷冻保存 胚胎;然而，我们目前没有斑马鱼胚胎冷冻保存的工作方案。尽管最近 优化，精子冷冻保存-目前的种质保存方法-只保存男性- 衍生基因组尽管冷冻保存的单倍体父本基因组随时间保持稳定， 用于提供雌性的活种群的变化不断累积。因此，胚胎的基因组有一半与 原来的鱼，一半不是。 我们将开发一个完整的平台，为四个斑马鱼二倍体种质资源保存 参考菌株。这个平台将有助于保存整个基因组接近已公布的序列信息 或新的序列，当它们变得可用时。为了实现这一目标，我们在密歇根州立大学的实验室 密歇根大学（MSU）将与水生种质和遗传资源中心（AGGRC）合作， 路易斯安那州立大学农业中心（LSUAC）和斑马鱼国际资源中心（ZIRC） 在俄勒冈州大学。新的二倍体种质保存综合平台包括两个 关键步骤：1）二倍体体细胞的分离、培养和冻存，2）细胞的解冻和使用 他们通过体细胞核转移（SCNT -克隆）获得原始菌株，这是我们拥有的一种技术， 成功地应用于生产创始人个人。该方法保留了所有等位基因的序列， 基因组该项目的具体目标是：目标1。开发一个综合平台来分离，培养， 冷冻保存并对来自野生型系的二倍体细胞进行基因分型。目标二。斑马鱼克隆的标准化 程序，包括纯合斑马鱼系的产生。目标3.宣传、传播和 使用二倍体体细胞保存方法培训资源中心和实验室人员。在 在这个R24提案结束后，科学界将获得：1）最稳定的二倍体基因组 使用了精子冷冻保存不可能使用的参考线，增加了精子冷冻保存的严格性和重现性。 实验室内和实验室之间的实验结果;和2）能够增强 研究细胞和器官移植，性别决定和其他方法。我们还将证明， 新的综合平台将能够：1）拯救濒临灭绝的宝贵研究路线， 只有一种性别的成年动物存活; 2）从被污染的管路中消除病原体。

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.