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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

基本信息

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
项目类别：
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

项目摘要

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