遗传操作新技术的建立及应用

Understanding the origin and fate of a particular type of cells in development and diseases is essential in stem cell biology and regenerative medicine. The current golden standard technology in revealing the fate of a cell in vivo is genetic lineage tracing, which is largely based on Cre-LoxP mediated recombination. Albeit powerful and useful in many cases, this technique has the inherited shortcoming that evokes disputes over the interpretation of the lineage tracing data in many fields. The specificity of Cre hinges on the gene promoter by which the Cre is driven. The resolution on expression map of Cre is not always as clear as “yes” or “no”, while, the fate, as the readout of the Cre-LoxP recombination, is binary “yes” or “no”. In some scenarios, there are discrepancies between the expression map and fate map, constituting a formidable technical barrier in the lineage tracing. Many important scientific questions regarding cellular origin and fate remain unsolved due to this technical hurdle. Our aim is to develop a new technique that is based on dual-recombination system for more accurate lineage tracing and also genetic targeting. This new system will not resolve the ectopic recombinase expression issue, but it could circumvent this hurdle by resolution of ectopic recombination and tracing. We would take advantage of this dual recombination system to resolve the major questions in cardiovascular field including the existence of cardiac stem cell in adult heart. In addition, the combination of Dre and Cre yields more precise genetic targeting in vivo, and we generated sequential intersectional genetics and provided an example of genetic targeting blood vessels in organ-specific manner, eg. targeting coronary vessels or brain vessels only. Due to the compatibility with previous Cre-LoxP recombination based system and openness by designing, this dual-recombination technology would aid in delineating lineage map and regulation of fate determination in stem cell biology and regenerative medicine.

研究体内特定类型（干）细胞的起源及命运，揭示（干）细胞在发育、疾病和组织再生中的转分化现象及细胞命运调控机制具有重大的科学意义。基于Cre-LoxP同源重组的传统遗传操作技术包括遗传示踪技术已经得到广泛应用，推动了细胞谱系的研究。然而这项技术本身存在一定的局限性，即引导Cre重组酶表达的基因并非完全特异性，从而导致了许多重大科学问题仍然存在争论。Dre-Rox同源重组系统在DNA重组上具有与Cre-LoxP同样的高效性和忠实性。我们利用二者之间相互排他性，将两种同源重组酶及其各自的识别位点在小鼠基因座位上有机地结合起来，根据两种同源重组系统不同的排列组合构建了一系列转基因小鼠。通过对心血管发育和再生中的谱系建立研究，举例证明如何利用新遗传操作技术示踪心肌干细胞以及在体精准遗传靶向。该遗传操作新技术的建立和应用有助于揭示（干）细胞在发育、疾病及再生中的起源、谱系建立及其分子调控机制。

基于Cre-LoxP同源重组的传统遗传操作技术已经得到广泛应用，但它存在着技术瓶颈，比如非特异性遗传靶向。从而导致了许多重大科学问题仍然存在争论。我们在这个项目中引入了新的Dre-Rox同源重组系统，它与Cre-LoxP具有同样的高效性和忠实性，这两个酶系统是正交体系。我们两种同源重组酶及其各自的识别位点在小鼠基因座位上通过一定的排列组合，构建一些列的工具小鼠。通过对心血管发育和再生中的关于心肌细胞的谱系建立研究，我们证明了成体心脏不存在心肌干细胞，发现新的心肌细胞主要来自与自我增殖，而非干细胞的分化。此外，我们还利用双同源重组酶系统实现了器官特异性遗传靶向，比如可以在心脏或大脑的内皮细胞特异表达的Cre工具小鼠。这些研究证明利用新遗传操作技术实现更为精准的细胞示踪和精准遗传靶向。该遗传操作新技术的建立和应用有助于揭示（干）细胞在发育、疾病及再生中的起源、谱系建立及其分子调控机制。