Development of genome-modified Xiphophorus

基因组修饰剑龙的开发

基本信息

批准号：
10689339
负责人：
Manfred Schartl
金额：
$ 18.81万
依托单位：
TEXAS STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10689339
关键词：
Acceleration Adopted Adult Animal Model Automobile Driving Backcrossings Behavioral Biological Biological Models Biomedical Research CRISPR/Cas technology Cancer Etiology Cancer-Predisposing Gene Candidate Disease Gene Cells Chronobiology Clustered Regularly Interspaced Short Palindromic Repeats Communities Compensation Complement Detection Development Diagnosis Disease Disease model Distant Elements Embryo Embryonic Development Engineering Environment Environmental Risk Factor Enzymes Exhibits Female Fishes Gene Transfer Genes Genetic Genetic Drift Genome Genomics Goals Guide RNA Health Heart Diseases High-Throughput Nucleotide Sequencing Human Hybrids Immunity Immunologics In Vitro Individual Institution Investigation Knock-out Laboratories Laboratory Animal Models Laboratory Organism Lentivirus Malignant Neoplasms Masks Medical Medical Research Metabolic Diseases Metabolism Methodology Methods Mission Modeling Modification Molecular Mus Mutation National Human Genome Research Institute National Institute of Allergy and Infectious Disease National Institute of Child Health and Human Development National Institute of Diabetes and Digestive and Kidney Diseases National Institute of Environmental Health Sciences National Institute of General Medical Sciences National Institute of Mental Health Obesity Optics Organism Oryziinae Outcome Oviparity Parasitology Phenotype Photobiology Physiological Physiological Processes Pigmentation physiologic function Pilot Projects Procedures Process Protocols documentation Rattus Regulation Research Resistance Rodent Model Severity of illness Skin Cancer Speed Supplementation Survival Rate System Targeted Research Techniques Technology Testing Therapeutic Toxicology Transgenic Animals Transgenic Organisms Translational Research United States National Institutes of Health Untranslated RNA Validation Work Xiphophorus Zebrafish anticancer research coping detoxication dietary early embryonic stage egg embryo culture experimental study gene environment interaction gene function genetic variant gonad development human disease human model improved innovation interest knockout gene lentivirally transduced melanoma mutant new technology reproductive sex determination technology development tool trait translational model translational study tumor progression viral DNA

项目摘要

Project Summary: Xiphophorus fishes are best known as long-standing biomedical models for translational research on cancer development, photobiology and metabolism. They are widely used in research topics that connect to the missions of multiple NIH Institutions/Centers. Xiphophorus have been used for behavioral (NIMH), evolutionary (NHGRI), physiological (NIGMS), developmental, toxicological (NIEHS), genomics (NHGRI), immunological (NIAID), and parasitological studies (NIAID), dietary effects on metabolism (NIDDK), sex determination and gonad development (NICHD), maturation (NICHD), chronobiology (NIGMS), cancer etiology (NCI), and optical genetic regulation (NIGMS). Xiphophorus fish exhibit several features that no other animal models share, including a high level of inter-species genetic divergence, the capability of producing inter-species and backcross hybrids, and adaptive phenotypes representing several human diseases. Xiphophorus is an evolutionary mutant model that is particularly suited to search for disease-modifying factors. Recent progress in high-throughput genomic methods has accelerated the detection of candidate disease-causing genes involved e.g. in modifying tumor progression, in obesity, heart diseases or immunity regulation. These and more genes await functional validation and molecular mechanism characterization to promote them to translational studies. Because Xiphophorus is a live-bearing fish species, transgenic technologies that have been developed in egg-laying model species cannot be utilized for Xiphophorus because such methods work only in the one-cell stage embryo, which cannot be isolated and cultured in Xiphophorus. Therefore, there is an urgent necessity to develop an alternative technique of modifying the genome of live-bearing fish for versatile applications. We propose as an innovative approach to use lentiviral transduction for gene transfer to late-stage Xiphophorus embryos, which can be cultured in vitro. To accomplish this, we will improve the in vitro culture conditions to a point where a sufficiently high number of genome- modified embryos will develop to fertile adults and give rise to stable genetically altered lines. The deliverable is a protocol that can be readily adopted by other laboratories and can be applied to other livebearing fish models. For the proof-of-concept, we will use the CRISPR/Cas-9 system to knock-out a Xiphophorus pigmentation-driving and cancer-predisposing gene, xmrk, that will result in an engineered fish that is resistant to crossing-conditioned melanoma. We will deliver a new Xiphophorus line that will be useful for studies on skin cancer and a protocol for genome modification of live-bearing fish. While Xiphophorus has since decades served mainly as a model in cancer research, and more recently also for obesity, the establishment of transgenic technologies will open the next chapter of research with this model and boost its usage for additional biomedical research through the possibility to generate new disease models by genome modification. The outcome of the proposal will enhance the use of the Xiphophorus model and ultimately lead to a better understanding of human disease with impacts on diagnosis and therapeutic strategies.

项目概要：剑鱼作为转化研究的长期生物医学模型而闻名关于癌症发展、光生物学和代谢。它们广泛应用于以下研究主题连接多个 NIH 机构/中心的任务。剑尾鱼已被用于行为 (NIMH)、进化 (NHGRI)、生理 (NIGMS)、发育、毒理学（NIEHS）、基因组学（NHGRI）、免疫学（NIAID）和寄生虫学研究（NIAID）、饮食对新陈代谢 (NIDDK)、性别决定和性腺发育 (NICHD)、成熟的影响（NICHD）、时间生物学（NIGMS）、癌症病因学（NCI）和光学遗传调控（NIGMS）。剑鱼表现出其他动物模型所不具备的几个特征，包括高水平的种间遗传差异、产生种间和回交杂交的能力，以及代表几种人类疾病的适应性表型。剑尾龙是一种进化突变模型这特别适合寻找疾病缓解因素。高通量研究的最新进展基因组方法加速了相关候选致病基因的检测，例如：在改变肥胖、心脏病或免疫调节中的肿瘤进展。这些以及更多的基因等待功能验证和分子机制表征以促进它们转化研究。由于剑尾鱼是一种活鱼，转基因技术已被广泛应用。在产卵模型物种中开发的方法不能用于剑尾鱼，因为这种方法有效仅存在于单细胞阶段胚胎，不能在剑尾鱼中分离和培养。所以，迫切需要开发一种修饰活体基因组的替代技术鱼的多种应用。我们建议使用慢病毒转导作为一种创新方法将基因转移到可以在体外培养的晚期剑尾鱼胚胎中。为了实现这一目标，我们将改善体外培养条件，达到足够高数量的基因组- 改造后的胚胎将发育成具有生育能力的成年个体，并产生稳定的基因改变品系。这可交付成果是一个可以很容易地被其他实验室采用并可以应用于其他实验室的协议活鱼模型。为了进行概念验证，我们将使用 CRISPR/Cas-9 系统来敲除剑尾鱼色素沉着驱动和癌症易感基因 xmrk，将导致转基因鱼对交叉条件黑色素瘤具有抵抗力。我们将交付新的剑尾龙对皮肤癌研究有用的生产线和活体基因组修饰方案鱼。虽然剑尾鱼几十年来主要作为癌症研究的模型，最近同样针对肥胖症，转基因技术的建立将开启新的篇章使用该模型进行研究，并通过可能性促进其在其他生物医学研究中的使用通过基因组修饰产生新的疾病模型。该提案的结果将增强使用Xiphophorus模型并最终导致更好地了解人类疾病对诊断和治疗策略的影响。