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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：转基因动物是农业生产的重要工具