利用CRISPR/Cas9技术构建CreERT2定点敲入Isl1基因小鼠模型及分析

Construction and Analysis of Cre ERT2 Knock-in Mouse for Genetic Labling of Isl1^+ Cardiac Progenitor Cells by CRISPR/Cas9 Technology

心肌祖细胞增殖和分化是心脏损伤后修复再生的基础,而Isl1被认为是心肌祖细胞的特异性标志。为了研究以及示踪Isl1+心肌祖细胞及其分化后代,该文尝试利用成簇规律间隔短回文重复序列CRISPR/Cas9系统,将Cre ERT2定点插入到小鼠Isl1内源基因启动子之后,建立了Cre ERT2基因敲入小鼠模型。通过与Rosa26-lox P-neo-lox P-lac Z小鼠(Rosa26-lac Z+)交配,获得Isl1-Cre ERT(KI)/Rosa26-lac Z+双杂合小鼠。经过基因型鉴定、组织表达谱测定和X-gal染色、冰冻切片和石蜡切片等方法,确认基因敲入小鼠的Cre ERT2表达在成年小鼠心脏窦房结、心脏神经节、主动脉弓和肺动脉根部,与文献报道的Isl1表达部位相同。该研究建立的模型可为研究心肌祖细胞的增殖和谱系示踪提供重要的模型。

Cardiomyocytes in adult mammals retain a limited ability to proliferate in response to specific stimuli, but little is known about the origin of proliferating cells. LIM-homeodomain transcription factor islet- 1 （Isl 1）-expressing cells are used to study the optimal endogenous progenitor cells in cardiac regeneration. Using the CRISPR/Cas9 genomic editing technology, we constructed Isll-CreERT2 knock-in mouse model which harboringan CreERT2 cassette down steam of the lsll promoter. We crossed Isll-CreERT2 males with Rosa26-1oxP-neo-loxP- LacZ transgenic reporter mice （Rosa26-1acZ＋） and analyzed Isll＋ positive cells by X-Gal staining. The results showed that Tamoxifen-Inducible Cre/loxP recombination, as depicted by blue cells, existed in heart sinoatrial node, cardiac ganglia, the aortic arch and pulmonary roots in adult mice. Isll expression profile was corresponding with previous research. By this work, we established Isll-CreERT2 knock-in mouse model successfully, and the model provides a useful tool for tracing cardiac progenitor cells in cardiac regeneration. This study will be helofulfor understanding and treating cardiovascular diseases, clinical medicine and sports rehabilitation medicine.