Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, Japan.
Optics and Imaging Facility, National Institute for Basic Biology, Okazaki, Japan.
Sci Rep. 2024 Oct 28;14(1):25696. doi: 10.1038/s41598-024-77458-8.
The crustacean Daphnia magna is an emerging model for ecological and toxicological genomics. However, the lack of methods for spatial and temporal control of gene expression has impaired the elucidation of molecular mechanisms underlying responses to environments in vivo. Here we report local activation of the hsp70 promoter-driven gene cassette in D. magna by the infrared laser-evoked gene operator (IR-LEGO), a method for heating the target cells with infrared irradiation. We identified the heat-inducible promoter upstream of the D. magna hsp70-A gene. Using this promoter, we generated a transgenic Daphnia harboring the heat-shock responsive GFP reporter gene and confirmed that the GFP gene responds to heat treatment not only in juveniles and adults but also in embryos. We collected embryos from the reporter line and irradiated four different regions of interest in the embryos: a proximal region of the third thoracic segment, a part of the midline, a second maxilla, and a distal region of the endopodite of the second antenna, all of which increased GFP fluorescence with an infrared laser. Our results suggest that the IR-LEGO method is useful for spatial and temporal control of gene expression and would advance the functional genomics in D. magna.
大型溞（Daphnia magna）是生态和毒理学基因组学的新兴模式生物。然而，缺乏时空控制基因表达的方法，限制了对体内环境响应的分子机制的阐明。本文报道了利用红外激光诱导基因操作器（IR-LEGO）在大型溞中局部激活 hsp70 启动子驱动的基因盒，这是一种利用红外辐射加热靶细胞的方法。作者鉴定了大型溞 hsp70-A 基因上游的热诱导启动子，并利用该启动子构建了携带热休克响应 GFP 报告基因的转基因大型溞，证实 GFP 基因不仅在幼体和成虫，而且在胚胎中对热处理有反应。作者从报告系收集胚胎，并在胚胎的四个不同感兴趣区域进行了照射：第三胸节的近端区域、中线的一部分、第二大颚和第二触角的内附肢的远端区域，所有这些区域的 GFP 荧光都随着红外激光的照射而增强。研究结果表明，IR-LEGO 方法可用于时空控制基因表达，将推进大型溞的功能基因组学研究。
