Establishment of Somatic Cell Nuclear Transfer as a Universal Platform for Cloning Marmosets

体细胞核移植作为克隆狨猴通用平台的建立

• 批准号: 10510648
• 负责人: Jung Eun Park
• 金额: $ 22.08万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10510648
• 关键词: Address; Anatomy; Aneuploidy; Animals; Antimitotic Agents; Biomedical Research; Birth; Brain; CADASIL; CRISPR/Cas technology; Callithrix; Callithrix jacchus jacchus; Cell Nucleus; Cell fusion; Characteristics; Chemicals; Chromosomes; Chronic; Cloning; Cognitive; Combined Modality Therapy; Communities; Complex; Crossbreeding; Cytoplasm; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; DNMT3B gene; DNMT3a; Deacetylase; Development; Domestic Animals; Early Onset Alzheimer Disease; Embryo; Embryo Cloning; Embryo Transfer; Embryonic Development; Epigenetic Process; Female; Funding; Gene Expression; Gene Expression Profile; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic Engineering; Genetic study; Goals; Histone Acetylation; Histones; Human; In Vitro; Individual; Infant; Measures; Mechanics; Messenger RNA; Metabolic; Metaphase; Methods; Methyltransferase; Microvascular Dysfunction; Modeling; Mosaicism; Mus; Mutation; NOTCH3 gene; Neurosciences Research; Nuclear; Oocytes; Phenotype; Phylogenetic Analysis; Physiological; Pregnancy; Procedures; Process; Protocols documentation; RNA; Research; Research Support; Social Behavior; Somatic Cell; Stains; System; Techniques; Technology; Term Birth; Testing; Totipotent; Transgenes; Transgenic Organisms; Translational Research; Ultraviolet Rays; United States National Institutes of Health; Variant; Work; assistive reproductive technique; base; blastocyst; calcium indicator; epigenome; experience; genome editing; histone demethylase; human disease; human model; immune function; improved; inhibitor; interest; nervous system disorder; neuropsychiatric disorder; nonhuman primate; novel; nuclease; presenilin-1; preservation; prevent; reproductive; somatic cell nuclear transfer; tool; translational neuroscience

PROJECT SUMMARY/ABSTRACT The common marmoset (Callithrix jacchus) is a New World non-human primate (NHP) with several practical advantages in biomedical research. Due to their phylogenetic proximity, marmosets are a genetically diverse NHP species with similar physiological, metabolic, and immunological functions as humans. Marmosets retain the typical anatomical and functional organization of the human brain. Marmosets have complex cognitive and social behavior. The above characteristics place marmosets as an ideal NHP model to bridge the gap between mice and humans for both basic and translational neuroscience. Marmosets reach sexual maturity at circa 18 months and give birth to multiple infants twice a year. Their short gestation period and compatibility with gene editing techniques make marmosets ideally poised to become the NHP model of choice in studying the genetic causes of neurological and neuropsychiatric disorders and understanding brain function. We have successfully generated transgenic marmosets expressing genetically encoded calcium indicators and genetically engineered marmosets with NOTCH3 mutations that cause the small vessel disease CADASIL. We have also made marmosets harboring PSEN1 mutations that cause early-onset Alzheimer’s disease. Our gene-edited marmosets will enable us to investigate the genetic causes of chronic neurological disorders. However, two main issues hinder the broader availability of genetically engineered marmosets. First, lentiviral-based approaches suffer from an uncontrollable integration of transgene with variation in copy number, and nuclease-based gene editing produces embryos with mosaic editing, leading to unpredictable gene expression patterns and variable phenotype. Second, and most importantly, there are no efficient ways to propagate an individual showing the interest phenotype. This proposal addresses these shortcomings by developing and optimizing somatic cell nuclear transfer (SCNT) as a universal platform for cloning marmosets. We want to build on our vast experience generating genetically modified marmosets to (1) develop and optimize the enucleation procedure in marmoset somatic cell nuclear transfer and (2) develop and optimize approaches enabling technologies for efficient epigenetic reprogramming during somatic cell nuclear transfer in the marmoset. These results will establish optimized marmoset SCNT protocols and unravel a novel enabling universal platform to generate a sizeable number of cloned marmosets expressing any phenotype of interest. This platform will significantly facilitate the propagation and sharing of marmoset models for neuroscience and translational research supported by multiple NIH ICs.

项目总结/摘要 普通绒猴（Callithrix jacchus）是一种新大陆非人灵长类动物（NHP），具有几种实用的 生物医学研究的优势。由于它们的亲缘关系，绒猴是一种遗传多样性 NHP物种具有与人类相似的生理、代谢和免疫功能。绒猴保留 人脑的典型解剖学和功能组织。绒猴具有复杂的认知和 社会行为上述特征使绒猴成为理想的NHP模型，可以弥合以下两个方面的差距： 小鼠和人类的基础和转化神经科学。绒猴大约18岁达到性成熟 几个月，一年生两次多胞胎。它们的妊娠期短，与基因的相容性 编辑技术使绒猴成为研究遗传学的NHP模型的理想选择。 神经和神经精神疾病的原因和了解大脑功能。我们已经成功 产生表达遗传编码的钙指示剂的转基因绒猴， 具有NOTCH 3突变的绒猴，其导致小血管疾病CADASIL。我们也取得了 携带PSEN 1突变的绒猴导致早发性阿尔茨海默病。我们的基因编辑 绒猴将使我们能够调查慢性神经系统疾病的遗传原因。但是，两个主要 这些问题阻碍了基因工程绒猴的广泛应用。第一，基于慢病毒的方法 具有拷贝数变异转基因和基于核酸酶的基因的不可控制的整合 编辑产生具有马赛克编辑的胚胎，导致不可预测的基因表达模式和可变的基因表达模式。 表型第二，也是最重要的，没有有效的方法来传播一个表现出 感兴趣表型。该建议通过开发和优化体细胞来解决这些缺点 核移植（SCNT）作为克隆绒猴的通用平台。我们希望在我们丰富的经验基础上 产生遗传修饰的绒猴，以（1）开发和优化绒猴中的去核程序 体细胞核移植和（2）开发和优化方法，使技术能够有效地 绒猴体细胞核移植过程中的表观遗传重编程。这些结果将建立 优化的绒猴SCNT协议，并解开一个新的通用平台，以产生相当大的 表达任何感兴趣表型的克隆绒猴的数量。该平台将大大促进 传播和共享用于神经科学和转化研究的绒猴模型， NIH IC。