Direct generation of complex genetically-modified mouse models via embryonic stem cells

通过胚胎干细胞直接生成复杂的转基因小鼠模型

• 批准号: 10589018
• 负责人: Duancheng Wen
• 金额: $ 21.19万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589018
• 关键词: Acceleration; Alleles; Animal Model; Animals; Biological Models; Biology; Breeding; Bypass; CRISPR/Cas technology; Cattle; Cell Culture System; Cell Culture Techniques; Chimera organism; Complement; Complex; Consumption; Data; Development; Differentiation Inhibitor; ES Cell Line; Embryo; Epigenetic Process; Exploratory/Developmental Grant for Diagnostic Cancer Imaging; Female; Fostering; Generations; Genes; Genetic; Genetically Modified Animals; Genome; Genomic Instability; Goals; Knock-in; Knockout Mice; Lipids; MAPK3 gene; MEK inhibition; MEKs; Maintenance; Mediating; Medical Research; Meditation; Modification; Mouse Strains; Mus; Mutation; Pathway interactions; Process; Production; Public Health; Research; Research Personnel; Serum; Serum-Free Culture Media; Signal Pathway; Site; Speed; Stem cell pluripotency; System; Technology; Testing; Time; United States National Institutes of Health; Y chromosome deletion; conditional knockout; cost; embryonic stem cell; flexibility; genetic manipulation; high reward; high risk; human disease; improved; inducible Cre; inhibitor; interest; male; mouse model; mutant; novel; pluripotency; preservation; sex; stem cells; tool; transmission process

PROJECT SUMMARY: Genetically modified (GM) animals are essential tools for the study of both fundamental biology and human diseases. The production of GM animals relies on two critical technologies: 1) stable genetic modifications and 2) germline transmission of the mutations into a model system. A typical approach for creation of complex GM mice involves the generation of tetra-parental chimeras from normal embryos and GM embryonic stem (ES) cells, followed by multiple rounds of breeding to obtain both male and female mice for germline propagation. Two limitations dominate this approach. First, maintenance of pluripotency limits the complexity of genetic manipulations. Second, this process is time-consuming, laborious, and costly, particularly if the final objective requires many independent germline manipulations in the same animal. We propose a feasible strategy to accelerate the production of complex GM mouse models. Employing the technology of sex-reversion via CRISPR/Cas9-meditated Y chromosome deletion in male ES cells and our novel ES cell culture system, we can directly generate isogenic male and female mice from the same targeted ES cells through tetraploid complementation (4n). This strategy would bypass at least two mouse breeding generations: the chimera development step and the complex breeding process. We will target male (XY) ES cell lines for intended genetic alterations and follow the deletion of Y chromosome to generate monosomic XO female ES cells. Using this strategy, compound homozygous GM mouse strains could be established at unparalleled speed and costs. In this R21 application we propose the following two Aims: Aim 1: Optimize AX-based ES cell culture system for the production of GM mouse models. Aim 2. Direct generation of isogenic male and female complex GM mice using novel ES cell culture medium. If successful, our approach would have a great impact on GM mouse model construction in terms of versatility, speed, and cost. This ambitious endeavor to develop a breakthrough technology for creation of complex GM mouse models would also possibly foster novel research opportunities.

项目概述：转基因（GM）动物是人类发展的重要工具。 基础生物学和人类疾病的研究。转基因动物的生产 依赖于两项关键技术：1）稳定的遗传修饰和2）种系 将突变传递到模型系统中。一种典型的创建 复杂的转基因小鼠涉及从正常的四亲嵌合体的产生， 胚胎和转基因胚胎干细胞（ES细胞），然后进行多轮育种， 获得雄性和雌性小鼠用于生殖系繁殖。两个限制占主导地位 这种方法。首先，多能性的维持限制了遗传学的复杂性。 操纵第二，这个过程是费时、费力和昂贵的， 特别是如果最终目的需要许多独立的种系操作， 同样的动物。我们提出了一个可行的战略，以加快生产， 复杂的转基因小鼠模型。采用性逆转技术， CRISPR/Cas9介导的男性ES细胞中的Y染色体缺失和我们的新ES 细胞培养系统，我们可以直接从雄性小鼠和雌性小鼠产生同基因的 通过四倍体互补（4 n）相同的靶向ES细胞。这一战略将 绕过至少两代小鼠繁殖：嵌合体发育步骤， 复杂的繁殖过程。我们将靶向男性（XY）ES细胞系， 遗传改变并随后缺失Y染色体以产生单体XO 女性ES细胞使用这种策略，复合纯合GM小鼠品系可以 以无与伦比的速度和成本建立。在此R21应用中，我们提出 目的1：优化AX-ES细胞培养体系， 转基因小鼠模型。目标2.同基因雌雄复合体的直接产生 使用新型ES细胞培养基的GM小鼠。如果成功，我们的方法将有一个 在通用性、速度和成本方面对GM小鼠模型构建有很大影响。 这一雄心勃勃的奋进，以开发一种突破性的技术，创造复杂的 转基因小鼠模型也可能促进新的研究机会。