Correlating molecular behavioral phenotypes in a marmoset model of Huntingtons disease

摘要 常见的绒猴为理解人类的组织提供了一个非常相关的灵长类模型 神经系统和影响它的疾病。和人类一样，绒猴也表现出合作的社会性 行为，并具有高级认知过程，使他们在建模领域非常感兴趣 发育和精神疾病及其治疗。它们也是多代遗传的理想选择 因为它们一年生两次胎，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 在绒猴中，建立生殖系可传递的基因模型一直是该领域的一个挑战 记者和三核苷酸重复基因类似于他们的小鼠同行。效率极低的 灵长类动物中的同源重组(HR)已经排除了通过简单地注入 在体外受精(IVF)过程中，Cas9蛋白和引导RNA进入胚胎，就像创造基因敲除一样。 这一局限性阻碍了对亨廷顿氏症等更复杂的遗传神经系统疾病的建模 疾病(HD)和在绒猴中创造条件记者，这两者都是小鼠的支柱 神经遗传学领域。除了较低的HR频率外，造成敲打- INS包括直到最近还没有一个良好注释的绒猴基因组，缺乏推导 基态绒猴多能干细胞(CjPSCs)，绒猴受孕率低后 胚胎再植入，以及在绒猴领域普遍缺乏发育生物学专业知识。我们 建议利用我们实验室在发育生物学、试管受精技术和转基因干细胞方面的专业知识 生物学，以克服这一障碍，广泛使用绒猴。我们的目标是创造转基因敲入cjPSCs， 将它们转化为基态多能干细胞，然后将它们注射到试管受精桑椹胚中创建嵌合体 携带修改过的基因组的方正绒猴。我们的目标是从转基因配子中筛选出 方正绒猴创造F1后代，并用它们来关联 高清。作为原则证明，我们将重点关注三个敲入报告行，以广泛地针对兴奋性、抑制性、 和外周神经元群。如果成功，我们的目标将共同创造出第一个灵长类动物 用神经元特异性记者建立模型，将绒猴作为HD的有效模型，使其能够获得单一的- 灵长类疾病模型中HD早期细胞转录的变化，并最终与这些变化相关 分子水平会随着行为表型的变化而变化。这些目标将提供对生物学的基本见解 以及亨廷顿蛋白在不同类型神经元中的作用。该项目的成果还将 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, 我们产生的转基因绒猴将广泛应用于研究界，并使新的 研究神经回路、发育、行为和广泛的光遗传应用。

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.