STOCK Tg(Thy1-jRGECO1a)GP8.31Dkim/J

Stock number: 030526
Product name: Thy1-jRGECO1a-WPRE, line GP8.31
Research areas: Neurobiology Research, Research Tools

Description

These transgenic mice, made by the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project, express improved red fluorescent calcium indicator, jRGECO1a (derived from mApple), in subsets of cortical neurons and possibly other cell types and tissues.

Detailed description

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. This improved red GECI based on mApple (jRGECO1a) has sensitivity comparable to GCaMP6 green fluorescent calcium indicators. Red GECIs facilitate deep-tissue imaging, and dual-color imaging together with GFP-based reporters.

These transgenic reporter mice express jRGECO1a, an optimized red fluorescent GECI (Dana et al., eLIFE, 2016 - PMID 27011354), under the mouse Thy1 promoter. Expression has been examined in four cortical locations (the anterior lateral motor cortex, primary motor cortex, primary somatosensory cortex, and primary visual cortex), as well as the hippocampus. Cortical expression was comparable to adeno-associated virus (AAV) induced expression. Four jRGECO1a strains derived from the same transgenic vector have been made available to the JAX Repository (Founder line GP8.20, Stock No. 030525; founder line GP8.31, Stock No. 030526; founder line GP8.58, Stock No. 030527; and founder line GP8.62, Stock No. 030528 - April, 2017). Comparative data and images may be found at in an appended PDF.

For cortical imaging, founder line GP8.20 (see Stock No. 030525) shows denser labeling (~60% of cells) and founder line GP8.31 (this strain; Stock# 030526) shows sparser labeling (~30% of all cells). Both GP8.20 and GP8.31 both provide good signal levels across most of the cortex.

Red GECIs still suffer from several drawbacks compared to GCaMP6 sensors. The maximal fluorescence changes upon calcium binding are 3- to 4-fold less than the GCaMP6 indicators. This limits the overall dynamic range of red GECIs. The relatively low absorption cross-section of the red GECIs further reduces the achievable signal-to-noise ratio for in vivo imaging. Red GECIs show complex intracellular behavior, such as accumulation in lysosomes with long-term expression in the mouse brain. The presence of green fluorescence in all tested red GECIs suggests the existence of multiple protein species, some of which do not produce functional signals.

These mice were made by the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project at the HHMI's Janelia Research Campus.

Development

The mouse Thy1 promoter drives expression of this transgene that includes jRGECO1a (Dana et al., eLIFE, 2016 - PMID 27011354), nuclear export signal (NES) from protein kinase inhibitor alpha (PKIalpha), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and bovine growth hormone (bGH) polyadenylation (polyA) sequence.

The jRGECO1a feature is a modified form of R-GECO. R-GECO is derived from mApple (a red fluorescent monomer which is substantially brighter than mCherry and displays complex photoswitching behavior; see Shaner, 2008 PMCID PMC2853173) fused to calmodulin (CaM) and the CaM-interacting M13 peptide. In the presence of calcium, CaM undergoes a conformational change and associates with M13 to form a complex proximal to the chromophore inside the RFP β-barrel. The conformational change modifies the chromophore environment, modulating solvent access, chromophore K_a, absorption, and quantum yield, and altogether results in increased RFP brightness. Structure-guided mutagenesis and screening in a neuron-based assay has been successful in improving red GECI sensitivity and kinetics. The CaM region in jRGECO1a was modified with Q306D and M339F mutations. The M339F mutation accelerates kinetics, but does not affect response amplitude. Similar to other mApple-based indicators, jRGECO1a exhibits photoswitching in response to blue light.

Fluorescence has been tested under two photon imaging conditions with excitation of 1040-1100nm. See Dana, 2016 (PMID 27011354) for details.

The strain was created and maintained on a C57BL/6J genetic background by the donating laboratory.

This is founder line GP8.31.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Three of 27 markers throughout the genome were segregating from an unknown source (likely FVB). This suggests a contamination from an unknown source or incomplete backcross.

Allele

Symbol: Tg(Thy1-jRGECO1a)GP8.31Dkim
Name: transgene insertion GP8.31, Douglas Kim
MGI accession ID: MGI:5896540
Generation method: Transgenic
Attributes: Reporter
Synonyms: GP8.31
Marker symbol: Tg(Thy1-jRGECO1a)GP8.31Dkim
Marker name: transgene insertion GP8.31, Douglas Kim
Chromosome: UN
Strain of origin: C57BL/6J
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: Cortex
Molecular note: The mouse Thy1 promoter drives expression of this transgene that includes jRGECO1a, nuclear export signal (NES) from protein kinase inhibitor alpha (PKIalpha), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and bovine growth hormone (bGH) polyadenylation (polyA) sequence. jRGECO1a is a modified form of R-GECO. R-GECO is derived from mApple (a red fluorescent monomer which is substantially brighter than mCherry and displays complex photoswitching behavior) fused to calmodulin and the calmodulin binding region of chicken myosin light chain kinase (M13). The calmodulin region is modified with Q306D and M339F mutations. The M339F mutation accelerates kinetics, but does not affect response amplitude. For cortical imaging, founder line GP8.20 shows denser labeling (~60% of cells) and founder line GP8.31 shows sparser labeling (~30% of all cells). Both GP8.20 and GP8.31 both provide good signal levels across most of the cortex.