| Molecular Note | This transgene contains a calcium-sensing molecule, GCaMP2.2c, under the transcriptional control of the thymus cell antigen 1 (Thy1) promoter. The Thy1 promoter used here is a modified regulatory region of the "murine thy1.2 gene" (extending from the promoter to the intron following exon 4, excluding exon 3 and its flanking introns). The GCaMP2 consists of, from 5' to 3', a poly-histidine tag (6XHis), 13-residue peptide of myosin light chain kinase (M13), a circularly permutated eGFP interrupted at residue 145, calmodulin (CaM), and a polyA recognition sequence. Mutations resulting in increased brightness (D180Y and V93I), thermal stability (V163A, and S175G) and GFP dimerization prevention (A206K) were introduced. To further stabilize GCaMP2 and increase fluorescence, two more mutations (R2V and S118C) were introduced, resulting in the creation of GCaMP2.2c. Line 8 was generated.
The thymus cell antigen 1 (Thy1) promoter drives expression of an eGFP-calmodulin fusion protein, GCaMP2.2c in the central nervous system. Specifically, GCaMP is expressed in the cortex, hippocampus, thalamus, cerebellum, superior colliculus, amygdala, brain stem, retina and spinal cord. An increase of green fluorescence in specific neurons can be observed as the result of elevated intracellular calcium. In these GCaMP2.2c mice, cytoplasmic fluorescence is evident in apical dendrites, dendritic spines of layer V neurons of the living cortex. GCAMP expression is lower in these mice than in B6;CBA-Tg(Thy1-GCamp3)6Gfng/J mice (Stock No. 017893). GCaMP2.2c mice allow fluorescent detection of activated neurons at the cellular level in brain slices and live animals. |
