Application of Infrared Laser to the Zebrafish Vascular System Gene Induction, Tracing, and Ablation of Single Endothelial Cells
Application of Infrared Laser to the Zebrafish Vascular System Gene Induction, Tracing, and Ablation of Single Endothelial Cells
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DOI：
10.1161/atvbaha.112.300602
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发表时间：
2013-06-01
影响因子：
8.7
通讯作者：
Hitomi, Jiro
中科院分区：
文献类型：
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作者：
Kimura, Eiji;Deguchi, Tomonori;Hitomi, Jiro
目标红外激光诱发基因操纵子是一种新的显微方法，经过优化，可以在不造成光化学损伤的情况下加热活体生物体中的细胞。通过结合热休克反应的启动子系统，红外激光诱发基因操纵子能够在目标细胞中进行激光介导的基因诱导。我们将该方法应用于斑马鱼胚胎的血管系统，并证明其可用于研究体内血管形态发生的机制。 方法和结果-我们使用带有fli1：nEGFP的双转基因斑马鱼来识别内皮细胞，并用hsp：mCherry进行单细胞标记。通过优化照射条件，我们最终成功诱导单靶点内皮细胞表达mCherry基因，最高效率可达60%。此外，我们表明该系统可在某些条件下用于激光烧蚀。为了评估红外激光诱发基因操纵子，我们将该系统应用于第一节间动脉的内皮细胞，并捕获大脑和脊髓血管系统之间连接的图像。结论-我们的结果表明，红外激光诱发基因操纵子系统将通过标记或删除活体胚胎中的单个血管来控制单个内皮细胞中的时空基因激活，从而有助于阐明血管形态发生的机制。
Objective-Infrared laser-evoked gene operator is a new microscopic method optimized to heat cells in living organisms without causing photochemical damage. By combining the promoter system for the heat shock response, infrared laser-evoked gene operator enables laser-mediated gene induction in targeted cells. We applied this method to the vascular system in zebrafish embryos and demonstrated its usability to investigate mechanisms of vascular morphogenesis in vivo.Approach and Results-We used double-transgenic zebrafish with fli1:nEGFP to identify the endothelial cells, and with hsp:mCherry to carry out single-cell labeling. Optimizing the irradiation conditions, we finally succeeded in inducing the expression of the mCherry gene in single targeted endothelial cells, at a maximum efficiency rate of 60%. In addition, we indicated that this system could be used for laser ablation under certain conditions. To evaluate infrared laser-evoked gene operator, we applied this system to the endothelial cells of the first intersegmental arteries, and captured images of the connection between the vascular systems of the brain and spinal cord.Conclusions-Our results suggest that the infrared laser-evoked gene operator system will contribute to the elucidation of the mechanisms underlying vascular morphogenesis by controlling spatiotemporal gene activation in single endothelial cells, by labeling or deleting individual vessels in living embryos.