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). In mice of three high copy number transgenic lines (lines 11, 20 and 30) exhibiting preferential expression of erGAP1 in neural tissues, GFP fluorescence is first observed at embryonic days E12-E13 and is stable at least through 18 months. erGAP1 is strongly expressed in pyramidal CA1 and CA2 neurons and the dentate gyrus of the hippocampus, in the cortex, in both granule neurons and Purkinje cells of the cerebellum, and in dorsal root ganglion (DRG) neurons and the spinal cord. In all brain regions examined, erGAP1 is correctly targeted to the ER without evidence of aggregation. Its [Ca2+] sensor function in hippocampal and spinal sections and in individual neurons of various types isolated from these mice was demonstrated by reproducible, rapid, reversible changes in the F470/F405 fluorescence intensity in response to glutamate and caffeine at different concentrations. (J:206829)
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