Figure 1. Transgenic mice constructed with Cre-inducible, mitochondrially targeted GFP-aequorin.

(A) Schematic diagram showing the genetic design of transgenic animals. The transgene, pCAG-loxP-Stop-loxP-mtGA-PolyA can be activated by crossing mice with another mouse line expressing Cre under the control of different promoters for conditional activation of mtGA transcription. (B) Western blot on purified mitochondrial enriched fractions from skeletal muscle of transgenic mice crossed with PGK-Cre. Mitochondrial fractions were compared with transgenic mice, which were not expressing the mtGA protein and blots were developed with both aequorin and GFP antibodies. (C, D, E, F) Confocal analysis of mtGA fluorescence in anterior tibialis muscle fibers. (C and D) Direct fluorescence of GFP expression. (D) Enlargement of the frame area in C. (E and F) Overlay of anti-GFP labeling (green) and anti-cytochrome-C labeling (red), where yellow indicates co-localization of the two labels. (F) Enlargement of the frame area in E. Scale bars for C & E = 20 µm. Scale bars for D & F = 5 µm. (G–I). Gallery of post-embedding GFP immunogold electron micrographs from anterior tibialis mouse muscle. GFP is localized in mitochondria. GFP is visualized by sequential probing with anti-GFP antibody and IgG conjugated with 10 nm colloidal gold. Scale bars: G, 1 µm; H, 500 nm; I, 100 nm. (J) Ca²⁺ CRET activities on purified mitochondrial fractions from skeletal muscle, brain, heart and cellular extracts. CRET measurements are expressed as the ratio of green (515 nm) over blue (460 nm). (K) Schematic diagram of the GA-CLZN light reaction. The binding of Ca²⁺-ions to aequorin leads to a conformational change, which results in the oxidation of its bound substrate chromophore, coelenterazine. Non-radiative energy transfer then occurs from the excited state chromophore to GFP, which then emits light in the green (λmax = 510 nm).