Tools for gene editing in marmosets

用于绒猴基因编辑的工具 摘要 该提案的目标是创建一套高效的新工具和技术，用于生成 适合用作脑生理和功能模型的转基因绒猴。发展中的 在该物种中进行基因操作的有效方法将需要改进基因组编辑技术， 育种技术、卵巢刺激和取卵、胚胎培养和植入前筛选 以获得所需的基因变化。一大群绒猴是必要的，还有一只 完善技术的基础设施和拥有绒猴护理专业知识的跨学科团队，得到协助 繁殖技术，使用CRE驱动程序和CRISPR技术的组织特异性表达，以及行为 测试和神经精神分析。这项建议的具体目标是：(1)提高智能手机的技术 恒河猴的体外遗传操作，探索恒河猴基因编辑的新方法；(2)确定安全 利用启动子进行无处不在的表达，建立Cas9、Cre和GCaMP绒猴品系， 脑内选择性表达，或区域或细胞类型特异性表达；以及(3)产生绒猴模型 与人类大脑疾病的神经生物学相关的基因改变。这些研究将提供工具 在未来的绒猴基因编辑实验中，将建立一套初步的转基因工具线，用于 在野外使用，并将为基因改变的绒猴模型提供初步的原则证明线 可能与人类大脑疾病的神经生物学有关。

Tools for Gene Editing in Marmosets SUMMARY The goal of this proposal is to create an efficient new set of tools and techniques for the generation of genetically modified marmosets suitable for use as models of brain physiology and function. Development of efficient methods of genetic manipulation in the species will require refinement of genome editing technologies, breeding technologies, ovarian stimulation and oocyte retrieval, embryo culture, and pre-implantation screening of embryos for the desired genetic alternations. A large colony of marmosets will be necessary, along with an infrastructure to refine technologies and an interdisciplinary team with expertise in marmoset care, assisted reproduction technologies, tissue-specific expression with Cre-drivers and CRISPR technologies, and behavior testing and neuropsychiatric analysis. The Specific Aims of this proposal are to: (1) Improve technology for in vitro genetic manipulation of marmosets and explore novel methods for marmoset gene editing; (2) Identify safe harbor loci and establish Cas9, Cre, and GCaMP marmoset lines using promoters for ubiquitous expression, selective expression in brain, or regional or cell-type specific expression; and (3) Generate marmoset models with genetic alterations relevant to the neurobiology of human brain diseases. These studies will provide tools for future gene editing experiments in marmosets, will establish an initial set of genetically modified tool lines for use in the field, and will provide initial proof-of-principle lines for marmoset models of genetic alterations potentially relevant to the neurobiology of human brain diseases.