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

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。双 谱系追踪小鼠提供了关于正常发育过程中细胞命运表型转换的进一步信息。 发育、器官功能和疾病。这一提议旨在产生一系列新的独特的双重 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。 循环内皮细胞（EC）是血管生成和内皮细胞向间质细胞转化所必需的 （EndMT）参与疾病的纤维化。然而，描述成人EC的贡献， 重新进入细胞周期或进行EndMT仍然是一个挑战。尽管目前的谱系追踪小鼠 细胞，这些方法不能消融循环或经历EndMT的EC的特定亚群， 确定它们对疾病进展和严重程度的贡献。为了弥补这一知识差距，我们 将使用我们之前发表的连续直系同源DNA重组酶方法来微调DNA 在特定细胞中暂时重组。顺序DNA重组酶允许消融和 询问对器官功能的后续影响。将该策略应用于心肌细胞（CM）， 我们创造了一种新的转基因小鼠，它表达串联的正向DNA重组酶。我们 观察到消融内源性循环CM会使心肌梗死（MI）后的心脏功能恶化。 我们使用传统的小鼠转基因进行CM实验，这是一种费力、昂贵和耗时的方法， 消耗过程，需要超过12个月的时间才能获得研究所需的小鼠。然而，在这方面， “线性化dsDNA靶向整合”（TILD）-“规则间隔排列的 短回文重复序列”（CRISPR）提供了一种非常准确，高效和快速的替代方案， 产生转基因小鼠。因此，我们将合并CRISPR介导的基因组编辑和正向测序， DNA重组酶，以创造一个工具箱的下一代转基因小鼠，可以“混合和- 以研究体内EC循环和EndMT介导的纤维化。新的老鼠将扩大我们的 了解多种疾病中EC增殖和EndMT介导的纤维化，包括心脏病 纤维化、动脉粥样硬化、肺动脉高压、肝硬化和癌症。开发的方法 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。

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.