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.

项目概要 斑马鱼研究的严谨性和可重复性受到威胁，因为当前的存储方法 保存斑马鱼种质资源不足，这对于最常用的斑马鱼种质资源来说尤其成问题。 参考菌株如 AB、Tübingen (TU) 及其混合衍生物 SAT 和 NHGRI，用于 基因组编辑应用严重依赖于原始 DNA 的准确性。 四种参考菌株的序列是几年前在选定的个体中生成的，并且随机遗传 漂移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，尽管最近有斑马鱼胚胎冷冻保存的工作方案。 优化，精子冷冻保存——目前的种质保存方法——仅保存雄性 尽管冷冻保存的单倍体父本基因组随着时间的推移保持稳定，但基因组 变化在用于提供雌性的牲畜中积累，因此，胚胎的基因组有一半与母体相同。 原来的鱼，一半不是。 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 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) 俄勒冈大学的二倍体种质保护新综合平台包括两个部分。 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）获得原始菌株，这是我们拥有的技术 该方法成功应用于产生创始人个体，并保留了所有等位基因和的序列。 该项目的具体目标是： 目标 1. 开发一个集成平台来分离、培养、 冷冻保存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，包括纯合斑马鱼品系的产生。 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 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 细胞和移植器官、性别决定和其他方法的研究我们也将证明这一点。 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。