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：
10287090
负责人：
ALI H BRIVANLOU
金额：
$ 60.06万
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/10287090
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 Magnetic Resonance Imaging 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 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.

抽象的共同的marmoset提供了一个非常相关的私人模型，以理解人类的组织神经系统和影响它的疾病。像人类一样，摩尔马人也证明行为并具有先进的认知过程，使它们对建模的领域产生了极大的兴趣发育和精神病及其疗法。它们也是多代通用的理想选择实验每年两次出生，并且比大多数私人生成更快。但是， CRISPR/CAS9系统已用于敲除基因，并在可遗传的在摩尔果中的方式，在现场创建种系的基因模型是一个挑战记者和三核苷酸重复基因类似于其鼠。非常低的效率私人中的同源重组（HR）通过简单地注射就排除了敲入编码序列在体外受精过程（IVF）期间，CAS9蛋白和将RNA引导到胚胎中，如创建敲除所做的那样。这种局限性阻止了建模更普遍的复杂神经系统疾病，例如亨廷顿疾病（HD）和用于在Marmoset中创建有条件的记者，这两者都是小鼠的主要阶段神经遗传学领域。除了低HR频率外，其他障碍物创造了敲敲的种系传播 - INS包括缺乏注释良好的Marmoset基因组，直到最近，缺乏推导方案基态果果多能干细胞（CJPSC），在胚胎重新植入的胚胎，以及在Marmoset领域的发育生物学专业知识的普遍缺乏。我们提议利用我们实验室在发育生物学，IVF技术和转基因干细胞方面的专业知识生物学克服了这种障碍，以宽大地使用果胶。我们旨在创建转基因敲击CJPSC，将它们转换为基态多能干细胞，然后将它们注入IVF莫拉菌以形成嵌合携带改良基因组的创始人摩尔马斯群岛。然后，我们旨在将转基因游戏从创建的摩尔果会创建F1后代，并使用它们与分子行为表型相关联高清。作为原理证明，我们将专注于三个敲门记者线，以广泛靶向兴奋性，抑制性，和周围神经元种群。如果成功的话，我们的目标将导致创造第一个灵长类动物具有神经特异性记者的模型，建立Marmoset作为HD的有效模型，可以访问单个在原发性疾病模型中，HD早期的细胞转录组变化，并最终将其关联分子随行表型的变化。这些目标将为生物学提供基本的见解 HD和亨廷汀蛋白在不同类别的神经元中的作用。该项目的结果也将影响对影响人类的多谷氨酰胺神经退行性疾病的更好理解。此外，我们生成的转基因摩尔奶酪将广泛地用于研究社区，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。