Suzuki Motoshi, Toyoda Naoya, Takagi Shin
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.
PLoS One. 2014 Jan 20;9(1):e85783. doi: 10.1371/journal.pone.0085783. eCollection 2014.
Methods for turning on/off gene expression at the experimenter's discretion would be useful for various biological studies. Recently, we reported on a novel microscope system utilizing an infrared laser-evoked gene operator (IR-LEGO) designed for inducing heat shock response efficiently in targeted single cells in living organisms without cell damage, thereby driving expression of a transgene under the control of a heat shock promoter. Although the original IR-LEGO can be successfully used for gene induction, several limitations hinder its wider application. Here, using the nematode Caenorhabditis elegans (C. elegans) as a subject, we have made improvements in IR-LEGO. For better spatial control of heating, a pulsed irradiation method using an optical chopper was introduced. As a result, single cells of C. elegans embryos as early as the 2-cell stage and single neurons in ganglia can be induced to express genes selectively. In addition, the introduction of site-specific recombination systems to IR-LEGO enables the induction of gene expression controlled by constitutive and cell type-specific promoters. The strategies adopted here will be useful for future applications of IR-LEGO to other organisms.
能够让实验者自主控制基因表达开启或关闭的方法，对各种生物学研究都将十分有用。最近，我们报道了一种新型显微镜系统，它利用红外激光诱发基因操纵器（IR-LEGO），旨在在不损伤细胞的情况下，高效诱导活生物体中靶向单细胞产生热休克反应，从而驱动热休克启动子控制下的转基因表达。尽管原始的IR-LEGO能够成功用于基因诱导，但一些局限性阻碍了它的更广泛应用。在此，我们以线虫秀丽隐杆线虫（C. elegans）为研究对象，对IR-LEGO进行了改进。为了更好地进行加热的空间控制，引入了使用光学斩波器的脉冲照射方法。结果，早在二细胞期的秀丽隐杆线虫胚胎的单细胞以及神经节中的单个神经元都能够被诱导选择性地表达基因。此外，将位点特异性重组系统引入IR-LEGO，能够诱导由组成型和细胞类型特异性启动子控制的基因表达。这里采用的策略将对IR-LEGO未来应用于其他生物体有用。
