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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的普通狨猴为理解人类的组织提供了一个非常相关的灵长类动物模型与人类一样，狨猴也表现出合作性的社会性。行为并具有先进的认知过程，使他们对建模领域产生浓厚的兴趣它们也是多代遗传疾病的理想选择。实验中，它们每年生育两次，并且比大多数灵长类动物成熟得更快。 CRISPR/Cas9系统已被用来敲除基因并在可遗传的细胞中创建单个氨基酸的敲入在狨猴中，建立种系可遗传的基因模型一直是该领域的一个挑战生产者和三核苷酸重复基因的效率非常低。灵长类动物中的同源重组（HR）通过简单地注射就排除了敲入编码序列 Cas9 蛋白和引导 RNA 在体外受精 (IVF) 过程中进入胚胎，就像创建基因敲除一样。这种限制阻碍了对亨廷顿舞蹈症等更复杂的遗传神经系统疾病的建模疾病（HD）和在狨猴中创造条件生产者，这两者都是小鼠的支柱神经遗传学领域除了低HR频率外，还有其他障碍导致基因敲除的种系传播。 INS 包括直到最近还缺乏注释良好的狨猴基因组，缺乏衍生的协议基态狨猴多能干细胞（cjPSC），狨猴怀孕后的百分比较低胚胎再植入，以及狨猴领域发育生物学专业知识的普遍缺乏。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克服了狨猴广泛使用的障碍，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 桑葚中以创建嵌合体然后，我们的目标是从携带修饰基因组的创始人狨猴中筛选转基因配子。创始人狨猴创造了 F1 后代，并用它们来关联狨猴的分子行为表型 HD。作为原理验证，我们将重点关注三个敲入报告基因系，以广泛针对兴奋性、抑制性、如果成功的话，我们的目标将是创造出第一个灵长类动物。与神经元特异性生产者建立模型，将狨猴建立为有效的 HD 模型，能够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变化，并最终将这些变化关联起来这些目标将为生物学提供基本的见解。 HD 的研究以及亨廷顿蛋白在不同类别神经元中的作用也将是该项目的成果。影响更好地了解影响人类的多聚谷氨酰胺神经退行性疾病。我们产生的转基因狨猴将广泛提供给研究界，并启用新的研究神经回路、发育、行为和广泛的光遗传学应用。