Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
Laboratory of Bioresources, National Institute for Basic Biology, Okazaki, Aichi, Japan.
Genesis. 2024 Feb;62(1):e23519. doi: 10.1002/dvg.23519. Epub 2023 May 25.
Spatial and temporal control of transgene expression is a powerful approach to understand gene functions in specific cells and tissues. The Tet-On system is a robust tool for controlling transgene expression spatially and temporally; however, few studies have examined whether this system can be applied to postembryonic stages of Medaka (Oryzias latipes) or other fishes. Here, we first improved a basal promoter sequence on the donor vector for a nonhomologous end joining (NHEJ)-based knock-in (KI) system. Next, using transgenic Medaka for establishing the Tet-On system by KI, we demonstrated that doxycycline administration for four or more days by feeding can be a stable and efficient method to achieve expression of the transduced reporter gene in adult fish. From these analyses, we propose an optimized approach for a spatio-temporal gene-expression system in the adult stage of Medaka and other small fishes.
转基因表达的时空控制是理解特定细胞和组织中基因功能的一种强大方法。Tet-On 系统是一种用于空间和时间控制转基因表达的强大工具；然而，很少有研究探讨过该系统是否可以应用于斑马鱼（Oryzias latipes）或其他鱼类的胚胎后阶段。在这里，我们首先改进了供体载体上的基本启动子序列，用于基于非同源末端连接（NHEJ）的基因敲入（KI）系统。接下来，使用转基因斑马鱼通过 KI 建立 Tet-On 系统，我们证明了通过喂食给予多西环素四天或更长时间可以是一种稳定且高效的方法，可在成年鱼中实现转导报告基因的表达。通过这些分析，我们提出了一种在成年斑马鱼和其他小鱼中时空基因表达系统的优化方法。
