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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

基本信息

批准号：
10354630
负责人：
Duancheng Wen
金额：
$ 25.43万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-15 至 2024-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10354630
关键词：
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 Institutes Knock-in Lipids MAPK3 gene MEK inhibition MEKs Maintenance Mediating Medical Research Modification Mouse Strains Mus Mutation Pathway interactions Process Production Public Health Research Research Personnel Rewards Serum Serum-Free Culture Media Signal Pathway Site Speed Stem cell pluripotency System Technology Testing Time United States National Institutes of Health Y Chromosome Y chromosome deletion base conditional knockout cost embryonic stem cell flexibility genetic manipulation high reward high risk human disease improved inhibitor interest male mouse model mutant novel pluripotency preservation sex stem stem cells tool transmission process

项目摘要

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.

项目摘要：转基因 (GM) 动物是实现人类健康的重要工具研究基础生物学和人类疾病。转基因动物的生产依赖两项关键技术：1) 稳定的基因修饰和 2) 种系将突变传输到模型系统中。创建的典型方法复杂的转基因小鼠涉及从正常小鼠产生四亲嵌合体胚胎和转基因胚胎干（ES）细胞，然后经过多轮育种获得雄性和雌性小鼠进行种系繁殖。主要有两个限制这种方法。首先，多能性的维持限制了遗传的复杂性操纵。其次，这个过程费时、费力、成本高，特别是如果最终目标需要许多独立的种系操作同一种动物。我们提出了一个可行的策略来加速生产复杂的转基因小鼠模型。采用性别回复技术 CRISPR/Cas9介导的雄性ES细胞中Y染色体缺失和我们的新型ES 细胞培养系统，我们可以直接从细胞培养中产生同基因雄性和雌性小鼠通过四倍体互补 (4n) 获得相同的目标 ES 细胞。这一策略将绕过至少两代小鼠繁殖：嵌合体发育步骤和复杂的养殖过程。我们将针对雄性 (XY) ES 细胞系进行预期遗传改变并随着 Y 染色体的缺失产生单体 XO 雌性 ES 细胞。利用这种策略，复合纯合转基因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，我们建议以下两个目标：目标 1：优化基于 AX 的 ES 细胞培养系统用于生产转基因小鼠模型。目标2.直接产生同基因雄性和雌性复合体使用新型 ES 细胞培养基的转基因小鼠。如果成功的话，我们的方法将具有在通用性、速度和成本方面对转基因小鼠模型构建产生了巨大影响。这项雄心勃勃的努力旨在开发突破性技术来创造复杂的转基因小鼠模型也可能带来新的研究机会。