Tg(CAG-GFPuv/AEQ*)3Alns

Mice bearing this transgene express a fluorescent Ca2+ reporter protein that is targeted to the endoplasmic reticulum (ER). "GAP" is a fusion protein comprising a modified green fluorescent protein, GFPuv (GFP with amino acid substitutions Q80R, F99S, M153T, and V163A) joined by a 16-amino acid linker peptide (TATPATTPTTAPTAGT) to the Ca2+-sensing bioluminescent protein aequorin. To improve its performance in the ER, whose [Ca2+] is greater than cytosolic [Ca2+], its Ca2+ sensitivity was reduced by replacement of the acidic amino acid aspartate with alanine at three positions in the aequorin moiety (D117A, D119A, and D163A) to create "GAP1." Targeting of this ER-optimized Ca2+ sensor to the ER was achieved by appending the calreticulin signal peptide and the ER retention signal (KDEL) to its 5' and 3' termini, respectively, to generate "erGAP1." The construct encoding erGAP1 was cloned into the pCAGGS expression vector between the cytomegalovirus immediate early enhancer/chicken beta-actin promoter (CMV-IE/ACTB) and the rabbit beta-globin polyadenylation signal and 3'-untranslated region (3'-UTR). (J:206829)