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

建立斑马鱼二倍体种质保护综合平台

• 批准号: 10556907
• 负责人: JOSE B CIBELLI
• 金额: $ 75.39万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10556907
• 关键词: Address; Alleles; Animal Model; Biological Assay; Cell Separation; Cell Therapy; Cell Transplantation; Cells; Cloning; Collaborations; Communities; Cryopreservation; DNA Modification Process; DNA Sequence; Derivation procedure; Development; Diploid Cells; Diploidy; Disease; Embryo; Extinction; Female; Fishes; Funding; Generations; Genes; Genetic Drift; Genome; Genotype; Germ; Goals; Haploidy; Human Resources; Hybrids; Individual; International; Laboratories; Laboratory Personnel; Louisiana; Malignant Neoplasms; Methods; Michigan; Modeling; National Human Genome Research Institute; Oregon; Organ Transplantation; Output; Preparation; Procedures; Process; Protocols documentation; Publishing; Quality Control; R24; Replacement Therapy; Reproducibility; Research; Resources; Sampling; Scientist; Shipping; Somatic Cell; Standardization; Techniques; Time; Toxicology; Training; United States National Institutes of Health; Universities; Work; Zebrafish; agricultural center; animal cloning; autism spectrum disorder; cell type; cost; developmental genetics; egg; embryo cryopreservation; established cell line; experimental study; gene function; gene therapy; genetic resource; genome editing; genomic locus; human disease; human model; laboratory experience; male; mature animal; mutant; pathogen; preservation; quality assurance; sex; sex determination; somatic cell nuclear transfer; sperm cryopreservation; whole genome; zebrafish genome

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.

项目总结 斑马鱼研究的严密性和重复性处于危险之中，因为目前的储存方法 保存斑马鱼种质资源不足。这对于使用最频繁的 参考菌株，如AB，Tübingen(TU)及其杂交衍生物SAT和NHGRI，用于 基因组编辑应用程序在很大程度上依赖于序列信息的准确性。原始的DNA 这四个参考菌株的序列是几年前在选定的个体中产生的，并随机遗传 漂移逐渐修改了这些基因组序列。其他动物模型依赖于超低温保存 然而，我们目前还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-目前种质保存的方法-只保存男性- 衍生的基因组。尽管冷冻保存的父本单倍体基因组随着时间的推移保持稳定，但基因组 变化累积在用于提供雌性的活体种群中。因此，胚胎的一半基因组与 原来的鱼，一半不是。 我们将为这四个物种开发一个斑马鱼二倍体种质资源保护的综合平台 参比菌株。这个平台将有助于保存整个基因组，使其接近已公布的序列信息 或新的序列，当它们可用时。为了实现这一目标，我们在密歇根州立大学的实验室 密歇根州立大学(MSU)将与水产种质和遗传资源中心(AGGRC)在 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) 在俄勒冈大学。新的二倍体种质资源保存综合平台包括两个 关键步骤：1)二倍体细胞的分离、培养和超低温保存；2)细胞的解冻和利用 通过体细胞核移植(SCNT-克隆)获得原始菌株，这是我们拥有的一种技术 成功申请产生创客个人。这种方法保留了所有等位基因的序列，并 基因组。该项目的具体目标是：目标1.开发一个综合平台，以隔离、培养、 野生型二倍体细胞的超低温保存及基因分型。目的2.斑马鱼克隆的标准化 程序，包括纯合斑马鱼品系的产生。目标3.推广、传播和 利用二倍体体细胞保存法对资源中心和实验室人员进行培训。在 在这份R24提案结束后，科学界将获得：1)稳定的二倍体大多数基因组 使用参照系，这是冷冻精子不可能的，增加了精子的严密性和重复性 实验室内和实验室之间的实验结果；以及2)能够增强 研究细胞和器官移植、性别决定和其他方法。我们还将展示这一点 新的集成平台将能够：1)在以下情况下拯救濒临灭绝的宝贵研究线路 只有一种性别的成年动物存活，以及2)从受污染的品系中消除病原体。