Genetically Engineered Zebrafish Biosensors

The fabp10-GRAB_ATP and fabp10-GRAB_Ado plasmids were constructed using PCR with KOD plus Neo (Toyobo) and In-Fusion Snap Assembly Master Mix (Takara) to produce vectors with Tol2 transposon sites. For fabp10-GRAB_ATP/-GRAB_Ado, the promoter sequence for hepatocytes was amplified using PCR and used to replace elavl3-GRAB_ATP/-GRAB_Ado. Multisite Gateway cloning was performed with the destination vector pDestTol2, the 5′-entry vector containing the fabp10 promoter, the middle entry vector containing pME-mCherry, and the 3′-entry vector containing p3E-polyA. Fabp10:mCherry was used to create fabp10 promoter elements. The zebrafish strains used were as follows: wild-type (AB strain; ZFIN, Eugen, OR, USA), fabp10-GRAB_ATP, and fabp10-GRAB_Ado. The plasmid was injected along with the transposon into the one-cell-stage embryo of wild-type zebrafish, as described previously^{49 (link)}. The plasmid insertion was confirmed by observing heart mCherry in 2 dpf larvae. Injected embryos were raised, and adult zebrafish 2 mpf were identified by amplifying the EGFP gene using PCR with the primers listed in Table 1. They were then outcrossed with wild-type zebrafish to obtain the next generation. Further, to confirm GFP expression in GRAB zebrafish, GRAB_ATP and GRAB_Ado larvae were embedded in E3 medium with ATP (5 mM) or Ado (6 mM) solutions. Live images were captured as described below.