A CRISPR-based modular transgenic system to advance in vivo investigations of angiogenesis and fibrosis

基于 CRISPR 的模块化转基因系统可推进血管生成和纤维化的体内研究

• 批准号: 10408193
• 负责人: Matthew J Wolf
• 金额: $ 24.23万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10408193
• 关键词: Ablation; Address; Adult; Aging; Animal Model; Atherosclerosis; Biomedical Research; Blood Vessels; CRISPR/Cas technology; Cardiac Myocytes; Categories; Cell Cycle; Cell Line; Cell Proliferation; Cells; Cirrhosis; Clustered Regularly Interspaced Short Palindromic Repeats; Consumption; DNA; DNA cassette; Development; Disease; Disease Progression; Disease model; Endothelial Cells; Endothelium; Fibrosis; Funding Opportunities; Gene Transfer Techniques; Genetic Recombination; Growth; Influentials; Institutes; Investigation; Knowledge; Label; Malignant Neoplasms; Mediating; Mesenchymal; Methods; Modeling; Mus; Myocardial Infarction; Myofibroblast; Organ; Outcome; Phenotype; Process; Proliferating; Publishing; Reporter; Research; Series; Severity of illness; System; Tamoxifen; Technology; Testing; Time; Tissues; Transgenic Animals; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; angiogenesis; base; cadherin 5; coronary fibrosis; ds-DNA; experimental study; genetic manipulation; genome editing; heart function; high reward; high risk; in vivo; interest; mouse model; next generation; periostin; pulmonary arterial hypertension; recombinase; single cell sequencing

Transgenic lineage-tracing mice are among the most influential biomedical research technologies. Dual lineage-tracing mice provide further information on phenotypic switching of cell fates during normal development, organ function, and disease. This proposal seeks to generate a series of new unique dual lineage-tracing transgenic mice to investigate angiogenesis and fibrosis in multiple disease models. Cycling endothelial cell (EC) are required for angiogenesis and endothelial-to-mesenchymal transition (EndMT) participates in fibrosis of disease. However, delineating the contributions of adult ECs that reenter the cell cycle or undergo EndMT remains a challenge. Although current lineage-tracing mice label cells, these approaches cannot ablate specific subpopulations of ECs that cycle or undergo EndMT to determine their contributions to disease progression and severity. To address this knowledge gap, we will use our previously published methods of sequential orthologous DNA recombinases to fine-tune DNA recombination temporally in specific cells. Sequential DNA recombinases allow the ablation and interrogation of subsequent effects on organ function. Applying this strategy to cardiomyocytes (CMs), we created a new transgenic mouse that expressed tandem orthologous DNA recombinases. We observed that ablating endogenous cycling CMs worsened heart function after myocardial infarction (MI). We used conventional mouse transgenesis for the CM experiments, a laborious, expensive, and time- consuming process, taking over twelve months to obtain mice needed for investigations. However, advances in “Targeted Integration with Linearized dsDNA” (TILD) - “Clustered Regularly Interspaced Short Palindromic Repeats” (CRISPR) provide a remarkably accurate, efficient, and rapid alternative to generate transgenic mice. Therefore, we will merge CRISPR-mediated genome editing and orthologous DNA recombinases to create a toolbox of next-generation transgenic mice that can be “mixed-and- matched” to investigate EC cycling and EndMT-mediated fibrosis in vivo. The new mice will expand our knowledge of EC proliferation and EndMT-mediated fibrosis in multiple diseases, including cardiac fibrosis, atherosclerosis, pulmonary arterial hypertension, cirrhosis, and cancer. The methods developed will enable the rapid creation of various transgenic mice that use sequential DNA recombinases to restrict Cre expression and investigate cell cycling and cell fate switching in vivo.

转基因谱系追踪小鼠是最具影响力的生物医学研究技术之一。 谱系追踪小鼠提供了正常情况下细胞命运表型转换的进一步信息 该提案旨在产生一系列新的独特的双重因素。 谱系追踪转基因小鼠研究多种疾病模型中的血管生成和纤维化。 血管生成和内皮间质转化需要循环内皮细胞 (EC) (EndMT) 参与疾病的纤维化，然而，描述了成人 EC 的贡献。 尽管目前的谱系追踪小鼠标签，重新进入细胞周期或进行 EndMT 仍然是一个挑战。 细胞，这些方法不能消除循环经历或 EndMT 的特定 EC 亚群 确定它们对疾病进展和严重程度的贡献为了解决这一知识差距，我们 将使用我们之前发布的连续直系同源 DNA 重组酶方法来微调 DNA 顺序 DNA 重组酶允许在特定细胞中进行暂时的消融和重组。 将该策略应用于心肌细胞（CM），探讨对器官功能的后续影响。 我们创造了一种表达串联直系同源 DNA 重组酶的新转基因小鼠。 观察到消除内源性循环 CM 会使心肌梗死 (MI) 后的心功能恶化。 我们使用传统的小鼠转基因进行 CM 实验，这是一项费力、昂贵且耗时的工作。 然而，获得研究所需的小鼠的消耗过程需要十二个月以上。 “线性化 dsDNA 的靶向整合”（TILD）方面的进展 - “簇状规则间隔 “短回文重复”（CRISPR）提供了一种极其准确、高效和快速的替代方案 因此，我们将融合 CRISPR 介导的基因组编辑和直系同源小鼠。 DNA重组酶创建了下一代转基因小鼠的工具箱，可以“混合-并- 匹配”来研究 EC 循环和 EndMT 介导的体内纤维化。新小鼠将扩展我们的研究。 对多种疾病（包括心脏病）中 EC 增殖和 EndMT 介导的纤维化的了解 纤维化、动脉粥样硬化、肺动脉高压、肝硬化和癌症的方法。 将能够快速创建各种转基因小鼠，这些小鼠使用顺序 DNA 重组酶来限制 Cre 表达并研究体内细胞周期和细胞命运转换。