Establishing an Integrated Platform for Diploid Germplasm Conservation in Zebrafish (Danio rerio) – A&R

建立斑马鱼（Danio rerio）二倍体种质保护综合平台 – A

• 批准号: 10808534
• 负责人: JOSE B CIBELLI
• 金额: $ 27.25万
• 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: 2023-05-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10808534
• 关键词: Address; Administrative Supplement; Alleles; Animal Model; Award; Cell Separation; Cell Therapy; Cell Transplantation; Cells; Cloning; Collaborations; Communities; Cryopreservation; DNA Modification Process; DNA Sequence; Derivation procedure; Development; Devices; Diploid Cells; Diploidy; Disease; Embryo; Equipment; Extinction; Female; Fishes; Funding; Generations; Genes; Genetic Drift; Genome; Genotype; Germ; Goals; Grant; Haploidy; Hybrids; Individual; International; Laboratories; Laboratory Personnel; Louisiana; Malignant Neoplasms; Marketing; Methods; Michigan; Micromanipulation; Modeling; National Human Genome Research Institute; Oregon; Organ Transplantation; Parents; Procedures; Protocols documentation; Publishing; R24; Replacement Therapy; Reproducibility; Research; Resources; Scientist; Somatic Cell; Standardization; Techniques; Time; Toxicology; Training; United States National Institutes of Health; Universities; Work; Zebrafish; agricultural center; autism spectrum disorder; cost; developmental genetics; egg; embryo cryopreservation; gene function; gene therapy; genetic resource; genome editing; human disease; human model; instrument; laboratory equipment; male; mature animal; pathogen; preservation; sex; sex determination; somatic cell nuclear transfer; sperm cryopreservation; whole genome; zebrafish genome

This is the abstract of the parent 1R24OD034058-01 award. 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.

这是母奖 1R24OD034058-01 的摘要。斑马鱼的严谨性和重现性 由于目前保存斑马鱼种质的储存方法不充分，研究面临着危险。 这对于最常用的参考菌株（例如 AB、Tübingen (TU) 和 他们的混合衍生物 SAT 和 NHGRI，用于关键依赖于基因组编辑应用 影响序列信息的准确性。四种参考菌株的原始 DNA 序列为 几年前在选定的个体中产生，随机遗传漂变逐渐改变了这些基因组 序列。其他动物模型依赖于胚胎的冷冻保存。然而，我们目前还没有工作 斑马鱼胚胎冷冻保存方案。尽管最近进行了优化，精子冷冻保存—— 目前的种质保存方法——仅保存雄性基因组。虽然 冷冻保存的单倍体父本基因组随着时间的推移保持稳定，基因组变化在家畜中积累 用于提供女性。因此，胚胎的基因组一半与原始鱼相同，一半则不同。 我们将开发一个斑马鱼二倍体种质资源保护的综合平台，用于四个 参考菌株。该平台将有助于保存接近已发布序列信息的整个基因组 或新序列可用时。为了实现这一目标，我们密歇根州立大学的实验室 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) 在俄勒冈大学。新的二倍体种质资源保护综合平台包括两个 关键步骤：1) 二倍体体细胞的分离、培养和冷冻保存，2) 细胞解冻和使用 他们通过体细胞核移植（SCNT – 克隆）衍生原始菌株，这是我们拥有的技术 成功应用于产生创始人个人。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. 开发一个集成平台来隔离、培养、 冷冻保存来自野生型细胞系的二倍体细胞并对其进行基因分型。目标 2. 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. 推广、传播和 使用二倍体体细胞保存方法对资源中心和实验室人员进行培训。在 在 R24 提案结束后，科学界将获得：1）大多数物种的稳定二倍体基因组。 使用精子冷冻保存无法实现的参考线，提高了精子冷冻保存的严谨性和可重复性 实验室内部和实验室之间的实验结果； 2) 能够增强的纯合斑马鱼 细胞和器官移植、性别决定和其他方法的研究。我们还将证明这 新的集成平台将能够：1）拯救濒临灭绝的宝贵研究线 只有一种性别的成年动物还活着，2) 消除受污染品系中的病原体。