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）动物是必需的工具研究基本生物学和人类疾病。转基因动物的生产依靠两种关键技术：1）稳定的遗传修饰和2）种系将突变传输到模型系统中。创建的典型方法复杂的GM小鼠涉及从正常产生四核酸的嵌合体胚胎和GM胚胎茎（ES）细胞，然后进行多轮繁殖获得雄性和雌性小鼠进行种系繁殖。两个局限性占主导地位这种方法。首先，多能维持限制了遗传的复杂性操纵。其次，这个过程是耗时，费力且昂贵的，特别是如果最终目标需要许多独立的种系操作同一只动物。我们提出了一种可行的策略，以加速生产复杂的GM鼠标模型。通过性逆转技术通过 CRISPR/CAS9中的Y染色体缺失在雄性ES细胞和我们的新型ES中细胞培养系统，我们可以直接产生来自通过四倍体互补（4N），相同的靶向ES细胞。这个策略会绕过至少两个小鼠育种一代：嵌合体开发步骤和复杂的繁殖过程。我们将针对预期的男性（XY）ES细胞系遗传改变并遵循Y染色体的缺失以产生单体XO 雌性ES细胞。使用此策略，复合纯合通用GM小鼠菌株可以以无与伦比的速度和成本建立。在此R21应用程序中，我们提出了以下两个目的：目标1：优化基于AX的ES细胞培养系统的生产 GM鼠标型号。目标2。直接生成等源性男性和女性复合物 GM小鼠使用新型ES细胞培养基。如果成功，我们的方法将有一个在多功能性，速度和成本方面，对GM鼠标模型构建产生了巨大影响。这项雄心勃勃的努力是开发一种创造复杂技术的突破性技术通用汽车鼠标模型也可能会促进新的研究机会。