Spatio‐temporal control of targeted gene expression in combination with CRISPR/Cas and Tet‐On systems in Medaka

Summary 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.