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.

项目摘要 斑马鱼研究中的严格和可重复性处于危险之中，因为当前的存储方法 保存斑马鱼种质不足。对于最常用的 参考菌株，例如AB，Tübingen（tu）及其混合衍生物SAT和NHGRI，用于 严重取决于序列信息的准确性的基因组编辑应用。原始DNA 几年前在某些个体中生成了四个参考菌株的序列，随机通用 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；但是，我们目前没有用于斑马鱼胚胎冷冻保存的工作协议。尽管最近 优化，精子冷冻保存 - 当前的种质保守方法 - 仅保留雄性 衍生的基因组。尽管冷冻保存的单倍体基因组随着时间的流逝，基因组保持稳定 用于提供女性的活股中积累的变化。那一半的胚胎基因组与 原始鱼，一半不是。 我们将开发一个用于斑马鱼中二倍体种质保护的集成平台 参考菌株。该平台将有助于保留靠近已发表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，我们在密歇根州的实验室 大学（MSU）将与水生的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。二倍体种质保护的新的集成平台包括两个 关键步骤：1）二倍体细胞的隔离，培养和冷冻保存，以及2）融化并使用 他们通过体细胞核转移（SCNT - 克隆）得出原始菌株，这是我们拥有的一种技术 成功地应用于创建者。此方法保留了所有等位基因的顺序和 基因组。该项目的具体目的是：目标1。开发一个隔离，文化， 来自野生型系的冷冻水果和基因型二倍体细胞。目标2。斑马鱼克隆的标准化 程序，包括纯合斑马鱼线的产生。目标3。促销，传播和 使用二倍体体细胞保护方法对资源中心和实验室人员进行培训。在 在此R24提案的结尾，科学界将可以使用：1）最稳定的二倍体基因组 使用的参考线，精子冷冻保存无法实现，增加了严格性和可重复性 实验室内和之间的实验结果； 2）能够增强的纯合斑马鱼 细胞和器官移植，性别确定和其他方法的研究。我们还将证明这一点 新的集成平台将能够：1）在扩展的边缘上营救宝贵的研究线 只有一种性别的成年动物仍然活着，2）从受污染的线条中消除病原体。