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.

项目摘要/摘要 常见的绒猴(Callithrix Jacchus)是一种新世界的非人灵长类动物，具有几种实用的 在生物医学研究方面的优势。由于它们在系统发育上的相似性，绒猴在遗传上是多样化的 具有与人类相似的生理、代谢和免疫功能的NHP物种。绒猴保留了 人脑的典型解剖和功能组织。绒猴有复杂的认知和 社交行为。上述特点使绒猴成为一种理想的NHP模型，以弥补 老鼠和人类的基础神经科学和翻译神经科学。绒猴在18岁左右达到性成熟 几个月，一年生两次多个婴儿。它们的妊娠期短，与基因的亲和性 编辑技术使绒猴成为研究基因的NHP模型的理想选择 神经和神经精神障碍的原因和对大脑功能的了解。我们已经成功地 产生的表达遗传编码的钙指示剂的转基因绒猴和基因工程 携带NOTCH3突变的绒猴会导致小血管疾病CADASIL。我们还制作了 携带PSEN1突变的绒猴会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疾病的遗传原因。然而，主要有两个 这些问题阻碍了转基因绒猴的更广泛获得。首先，基于慢病毒的方法 遭受拷贝数变异的转基因和基于核酸酶的基因的不可控的整合 编辑通过镶嵌编辑产生胚胎，导致不可预测的基因表达模式和变量 表型。其次，也是最重要的是，没有有效的方法来传播一个人展示 兴趣表型。该方案通过发展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。我们想要建立在我们丰富的经验基础上 产生转基因绒猴以(1)开发和优化绒猴去核程序 体细胞核移植和(2)开发和优化使能技术高效的方法 绒猴体细胞核移植过程中的表观遗传重编程。这些结果将确立 优化绒毛SCNT协议并解开一种新的使能通用平台产生相当大的 表达任何感兴趣表型的克隆绒猴的数量。该平台将极大地促进 多个支持的神经科学和翻译研究的绒猴模型的传播和共享 NIH IC。