Development of genome-modified Xiphophorus

项目摘要： 剑尾鱼是最有名的长期生物医学模型的转化研究 关于癌症发展、光生物学和新陈代谢。它们被广泛用于研究主题， 连接到多个NIH机构/中心的使命。剑尾鱼被用来 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 影响代谢（NIDDK）、性别决定和性腺发育（NICHD）、成熟 （NICHD）、时间生物学（NIGMS）、癌症病因学（NCI）和光学遗传调节（NIGMS）。 剑尾鱼表现出其他动物模型所没有的几个特征，包括高水平的 种间遗传分化，产生种间和回交杂交种的能力，以及 适应性表型代表了几种人类疾病。剑尾鱼是一种进化突变模型 特别适合于寻找疾病调节因子。高通量技术的最新进展 基因组学方法加速了涉及例如以下的候选致病基因的检测： 改变肿瘤进展，肥胖症，心脏病或免疫调节。这些和更多的基因 等待功能验证和分子机制表征，以促进其翻译 问题研究由于剑尾鱼是一种活的鱼类， 在产卵模式物种中开发的方法不能用于剑尾鱼， 仅在单细胞期胚胎中，这不能在剑尾鱼中分离和培养。因此，我们认为， 迫切需要开发一种替代技术来修改活产的基因组， 鱼的多功能应用。我们提出了一种创新的方法，使用慢病毒转导， 将基因转移到晚期剑尾鱼胚胎中，可以在体外培养。为了做到这一点，我们 将改善体外培养条件，使足够高数量的基因组- 经过改造的胚胎将发育成可育的成年体，并产生稳定的基因改造品系。的 可交付成果是一种可被其他实验室轻易采用并可应用于其他实验室的方案。 活体鱼类模型。对于概念验证，我们将使用CRISPR/Cas-9系统敲除 一种剑尾鱼色素驱动和癌症易感基因xmrk， 基因改造的鱼对交叉条件性黑色素瘤有抵抗力我们会给你一个新的剑尾鱼 这将有助于皮肤癌的研究和基因组修饰的协议活轴承 鱼.几十年来，剑尾鱼主要作为癌症研究的模型， 最近，对于肥胖症，转基因技术的建立将开启下一个篇章， 使用这种模型进行研究，并通过可能性将其用于其他生物医学研究， 通过基因组修饰来产生新的疾病模型。提案的结果将加强 使用剑尾鱼模型，并最终导致更好地了解人类疾病， 影响诊断和治疗策略。

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.