Tn(Tol2-CAG-AMPKAR-EV)#Kete

A pT2A derived vector containing the following sequences was microinjected together with Tol2 transposase mRNA into B6C3F1 fertilized eggs: minimal 5' Tol2 recognition sequence (first 200 bp), CAG promoter, LoxP site flanked tdKeima (tandem dimer Keima red fluorescent protein) gene cassette, AMPKAR-EV (AMP-activated protein kinase sensor), and minimal 3' Tol2 recognition sequence (last 175 bp). AMPKAR-EV contains DNA sequence for the following peptides: YPet (yellow fluorescent protein optimized for energy transfer), a spacer (Leu-Glu), the FHA1 domain (aa 241-382) of yeast Rad53, a spacer (Gly-Thr), the EV (extension for enhanced visualization by evading extraFRET) linker, a spacer (Ser-Gly), the substrate peptide (GSGEGSTKMRRVATLVDLGTGGSEL), a spacer (Gly-Gly-Arg), SECFP (super enhanced cyan fluorescent protein), a spacer (Ser-Arg), the nuclear export signal peptide of the HIV-1 Rev protein (LQLPPLERLTLD), and the SV40 poly(A) signal. The construct was inserted into the genome by the transposase and subsequent cre-mediated recombination removed the floxed sequence, allowing for the expression of the biosensor transgene. Only when the substrate peptide sequence is phosphorylated (by Prkaa1 or Prkaa2) does the FHA1 domain interact with the substrate sequence, bringing the two fluorescent proteins in close proximity, allowing for energy transfer from SECFP to YPet and thus generating yellow in stead of cyan fluorescence. (J:254233)