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Transgenic (Reporter) |
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Insertion
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Mutation details: This transgene contains a calcium-sensing molecule, GCaMP3, 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 GCaMP used here 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 GCaMP and increase fluorescence, four more mutations (R2V, M66K, T116V and N363D) were introduced, resulting in the creation of GCaMP3. Line 6 was generated.
The thymus cell antigen 1 (Thy1) promoter drives expression of an eGFP-calmodulin fusion protein, GCaMP3 in the central nervous system. Specifically, GCAMP3, is expressed in 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 GCaMP3 mice, cytoplasmic fluorescence is evident in the somata of layer V and layer II/III neurons of the living cortex, and in glomerular and mitral cell layers of the olfactory bulb. GCAMP expression is higher in these mice than in B6;CBA-Tg(Thy1-GCamp2.2c)8Gfng/J mice (Stock No. 017892). GCaMP3 mice allow fluorescent detection of activated neurons at the cellular level within the olfactory system.
(J:187952)
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