Correlating molecular behavioral phenotypes in a marmoset model of Huntingtons disease

亨廷顿病狨猴模型中分子行为表型的相关性

• 批准号: 10459516
• 负责人: ALI H BRIVANLOU
• 金额: $ 55.79万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10459516
• 关键词: Address; Affect; Alleles; Amino Acids; Animal Model; Animals; Behavior; Behavioral; Biology; Birth; Brain imaging; Breeding; CRISPR/Cas technology; Callithrix; Callithrix jacchus jacchus; Cell Line; Cells; Code; Cognitive deficits; Collaborations; Communities; Complex; Corpus striatum structure; DNA; Derivation procedure; Development; Developmental Biology; Disease; Disease Progression; Disease model; ES Cell Line; Embryo; Exons; Fertilization in Vitro; Frequencies; Functional disorder; Genes; Genetic; Genome; Germ Cells; Germ Lines; Grant; Guide RNA; Heritability; Human; Huntington Disease; Huntington gene; Huntington protein; Imaging technology; In Vitro; Knock-in; Knock-out; Laboratories; Mental disorders; Modeling; Molecular; Monitor; Morula; Mus; Mutation; Neurodegenerative Disorders; Neurons; Onset of illness; Outcome; Performance; Peripheral; Phenotype; Pluripotent Stem Cells; Population; Pre-Clinical Model; Pregnancy; Primate Diseases; Primates; Proteins; Protocols documentation; Reporter; Reporter Genes; Reproductive Technology; Research; Retrieval; Role; Serial Magnetic Resonance Imaging; Social Behavior; Stretching; Surgical Replantation; System; Technology; Transgenic Mice; Transgenic Organisms; Trinucleotide Repeats; Universities; Visuospatial; behavioral phenotyping; behavioral response; blastocyst; cognitive process; developmental disease; differentiation protocol; embryonic stem cell; experimental study; homologous recombination; insight; interest; knockout gene; molecular phenotype; mutant; nervous system disorder; neural circuit; neurogenetics; neurophysiology; nonhuman primate; optogenetics; polyglutamine neurodegenerative diseases; prevent; relating to nervous system; single-cell RNA sequencing; social deficits; stem cell biology; stem cells; transcriptomics; transmission process

ABSTRACT The common marmoset provides a very relevant primate model for understanding the organization of the human nervous system and the diseases that affect it. Like humans, marmosets also demonstrate cooperative social behavior and have advanced cognitive processes, making them of great interest in the field for modeling developmental and psychiatric diseases and their therapies. They are also ideal for multigenerational genetic experiments as they give birth twice a year and mature faster than most primates. However, while the CRISPR/Cas9 system has been used to knockout genes and create knock-ins of single amino acids in a heritable manner in marmosets, it has been a challenge in the field to create germline transmissible models of gene reporters and trinucleotide repeat genes analogous to their murine counterparts. The very low efficiency of homologous recombination (HR) in primates has precluded knocking-in coding sequences by simply injecting Cas9 protein and a guide RNA into embryos during in vitro fertilization (IVF) as is done for creating knockouts. This limitation has prevented modelling of more genetically complex neurological diseases such as Huntington’s disease (HD) and for creating conditional reporters in marmosets, both of which are mainstays in the mouse neurogenetics field. In addition to low HR frequency, other barriers to creating germline transmission of knock- ins include the absence of a well annotated marmoset genome until recently, lack of protocols for derivation of ground state marmoset pluripotent stem cells (cjPSCs), the low percentage of marmoset pregnancies after embryo reimplantation, and a general deficiency of developmental biology expertise in the marmoset field. We propose to harness our labs’ expertise in developmental biology, IVF technologies, and transgenic stem cell biology to overcome this barrier to widespread use of marmosets. We aim to create transgenic knock-in cjPSCs, convert them into ground-state pluripotent stem cells and then inject them into IVF morula to create a chimeric founder marmoset that carries the modified genome. We then aim to screen the transgenic gametes from the founder marmosets to create the F1 progeny and use them to correlate the molecular-behavioral phenotype of HD. As proof-of-principle, we will focus on three knock-in reporter lines to broadly target excitatory, inhibitory, and peripheral neuronal populations. Together, if successful, our aims will result in creation of the first primate model with neuron-specific reporters, establish the marmoset as a valid model of HD, enable access to single- cell transcriptomic changes at the early stages of HD in a primate disease model, and finally correlate these molecular changes with the behavioral phenotype. These aims will provide fundamental insights into the biology of HD and the role of huntingtin protein in different classes of neurons. The outcome of this project will also influence a better understanding of poly-glutamine neurodegenerative diseases that affect humans. In addition, the transgenic marmosets that we generate will be broadly available to the research community and enable new studies into neural circuits, development, behavior, and a wide range of optogenetic applications.

摘要 普通狨猴为理解人类的组织提供了非常相关的灵长类动物模型 和人类一样，绒猴也表现出合作的社会性， 行为和先进的认知过程，使他们在建模领域的极大兴趣 发育和精神疾病及其治疗。它们也是多代遗传的理想选择。 因为它们一年生两胎，比大多数灵长类动物成熟得更快。然而，虽然 CRISPR/Cas9系统已被用于敲除基因并在可遗传的基因组中产生单个氨基酸的敲入。 以这种方式在绒猴中，在该领域中创建基因的种系传播模型一直是一个挑战。 报告基因和三核苷酸重复基因类似于它们的鼠对应物。效率极低， 灵长类动物中的同源重组（HR）已经排除了通过简单地注射 Cas9蛋白和指导RNA在体外受精（IVF）过程中进入胚胎，就像创建敲除一样。 这种局限性阻碍了对更复杂的遗传性神经疾病（如亨廷顿病）的建模 疾病（HD）和在绒猴中创建条件性报告基因，这两者都是小鼠中的支柱 神经遗传学领域除了低HR频率，其他障碍，创造生殖系传播爆震- 这些缺陷包括直到最近还没有一个注释良好的绒猴基因组， 基础状态的绒猴多能干细胞（cjPSC）， 胚胎移植，以及发育生物学专业知识在绒猴领域的普遍缺乏。我们 我建议利用我们实验室在发育生物学、体外受精技术和转基因干细胞方面的专业知识 生物学来克服这一障碍，以广泛使用狨猴。我们的目标是创建转基因敲入cjPSC， 将它们转化为基态多能干细胞，然后将它们注射到IVF桑葚胚中以创造嵌合体 携带基因组改造的原始绒猴。然后，我们的目标是筛选转基因配子从 创始人绒猴创造F1后代，并使用它们来关联的分子行为表型， HD.作为原理证明，我们将关注三种敲入报告基因系，以广泛靶向兴奋性，抑制性， 和外周神经元群体。如果成功的话，我们的目标将创造出第一个灵长类动物 模型与神经元特异性的报告，建立作为一个有效的模型，HD的绒猴，使访问单- 在灵长类动物疾病模型中HD早期阶段的细胞转录组学变化，并最终将这些 行为表型的分子变化。这些目标将为生物学提供基本的见解， HD和亨廷顿蛋白在不同类别的神经元中的作用。该项目的成果还将 影响对影响人类的多聚谷氨酰胺神经退行性疾病的更好理解。此外，本发明还提供了一种方法， 我们生产的转基因绒猴将广泛提供给研究界， 研究神经回路，发育，行为和广泛的光遗传学应用。