B6J.Cg-Gt(ROSA)26Sor^{tm95.1(CAG-GCaMP6f)Hze}/MwarJ

Stock number: 028865
Product name: Ai95(RCL-GCaMP6f)-D (C57BL/6J) or Ai95D (C57BL/6J)
Research areas: Cancer Research, Cell Biology Research, Neurobiology Research, Research Tools

Description

Ai95(RCL-GCaMP6f)-D (also called Ai95D) mice are a Cre-dependent, fluorescent, calcium-indicator tool strain. Ai95D has a floxed-STOP cassette preventing transcription of the GCaMP6 fast variant calcium indicator (GCaMP6f; a detector of single neuronal action potentials with fast response kinetics). After Cre exposure, bright EGFP fluorescence is observed following calcium binding (such as neuronal activation).

Detailed description

Stock No. 028865 is the C57BL/6J-congenic Ai95D mouse line (Ai95D (C57BL/6J)). In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. The phenotype described below is for Ai95D mice on a mixed genetic background (Stock No. 024105). It should be noted that the phenotype of the C57BL/6J-congenic line could vary from that originally described for mice on other genetic backgrounds. We will modify the strain description if necessary as published results become available.

Ai95(RCL-GCaMP6f)-D mice (also called Ai95Δneo or Ai95D) harbor the Rosa-CAG-LSL-GCaMP6f::deltaNeo conditional allele, designed with a floxed-STOP cassette upstream of the GCaMP6 fast variant calcium indicator (GCaMP6f; see detailed description below). Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of GCaMP6f is prevented by STOP cassettes. After exposure to Cre recombinase, bright EGFP fluorescence is observed following calcium binding (such as neuronal activation).

Specifically, the donating investigator reports Ai95D mice have no reported levels of EGFP fluorescence prior to exposure to Cre recombinase. When Ai95D mice are bred with a Cre-driver line to create double transgenic animals (GCaMP6f+/Cre+), cells expressing Cre exhibit low EGFP fluorescence in the absence of calcium binding. Following calcium binding (such as neuronal activation), greatly increased EGFP fluorescence is observed in these cells.

The donating investigator also reports that following neuronal activation, the fluorescent intensity observed in double transgenic Ai95D mice (GCaMP6f+/Cre+) is less than that of the triple transgenic Ai93 system (GCaMP6f+/tTA+/Cre+ ; see Stock No. 024103). Although both systems utilize the same GCaMP6f gene, they differ in chromosomal location and Tet-inducibility.

Heterozygous Ai95D mice are viable and fertile with no reported gross physical or behavioral abnormalities. The Ai95D colony at The Jackson Laboratory is viable and fertile as homozygous (November 2017).

The donating investigator also reports that following neuronal activation, the fluorescent intensity observed in double transgenic Ai95D mice (Cre+/GCaMP6f+) is greater than that of the double transgenic Ai96 system using GCaMP6 slow variant calcium indicator (Cre+/GCaMP6s+ ; see Stock No. 024106). Although both systems utilize Cre-dependent GCaMP6/EGFP fusion genes, they differ in the kinetics and sensitivity of their GCaMP6 variants.

For characterization information of the Ai95 allele, see images at the Allen Institute for Brain Science website (Ai95(RCL-GCaMP6f) images).

The GCaMP6 fast variant calcium indicator (GCaMP6f) is a detector of single neuronal action potentials with fast response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. Compared to GCaMP6s, GCaMP6f has faster response but is less sensitive and faster to decay. In the absence of calcium binding, dim EGFP fluorescence is expected as there is a pore from the outside of its barrel into the chromophore. Upon calcium binding, this pore becomes occupied and fluorescence is increased.
The GCaMP6f fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). GCaMP6f has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with M153K, V163A, S175G, D180Y, T203V, A206K and V93I mutations to increase dynamic range/baseline fluorescence]), and a rat calmodulin DNA fragment (CaM; aa 2-148 with N60D, D78Y, T79R, S81T and R90G mutations [also called N362D, D380Y, T381R, S383T and R392G, respectively] to increase the fluorescence change for small calcium transients, as well the A15E (A317E) mutation for accelerated kinetics. The T302L and R303P amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function.

Development

The Ai95(RCL-GCaMP6f)-D allele (also called Ai95Δneo or Ai95D) was designed by Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a floxed-STOP cassette upstream of the GCaMP6 fast variant calcium indicator (GCaMP6f), all inserted into the Gt(ROSA)26Sor locus. Specific details below.

The Rosa-pCAG-LSL-GCaMP6f-WPRE-bGHpA-AttB-PGK-neo-polyA-AttP targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6f coding sequence (described below), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance mRNA transcript stability), a bovine growth hormone polyA signal, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, and an AttP site. This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were selected. Chimeric mice were bred to C57BL/6-congenic PhiC31-expressing mice (see Stock No. 007743) to remove the AttB/AttP-flanked PGK-Neo-polyA cassette and replace it with the recombined AttB/AttP site (AttL). The resulting Ai95D mice were bred to C57BL/6J wildtype mice for at least one additional generation (and the PhiC31 gene was removed). In 2014, Ai95D heterozygous males on a mixed genetic background (estimated 75-85% C57BL/6 ; with black coat color) were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. An aliquot of that frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664) and rederive our live colony as Stock No. 024105, which was thereafter maintained by breeding heterozygous mice with wildtype mice from the colony or with C57BL/6J inbred mice.
In 2015, Dr. Melissa R. Warden (Cornell University) purchased some Stock No. 024105 animals (total backcross generation N2-4 onto C57BL/6J). They further backcrossed them to C57BL/6J inbred mice for five additional generations. The resulting C57BL/6J-congenic strain, called Ai95(RCL-GCaMP6f)-D (C57BL/6J) or Ai95D (C57BL/6J), was sent to The Jackson Laboratory Repository in 2016 as Stock No. 028865. Dr. Warden's lab reports that during backcrossing the Y chromosome was made to be C57BL/6J origin. Upon arrival, males were used to cryopreserve sperm. To establish a living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664).
In 2017, a 153 SNP analysis (with markers covering all 19 chromosomes and the X chromosome) was performed on the first generation of rederived mice at The Jackson Laboratory Repository (heterozygous for the Ai95D allele). This showed all markers as C57BL/6J allele-type with only one exception - the first marker distal to the targeted Gt(ROSA)26Sor locus was segregating with the Ai95D allele (i.e., heterozygous for C57BL/6J;129 allele-type).

The GCaMP6 fast variant calcium indicator (GCaMP6f) is a detector of single neuronal action potentials with fast response kinetics (Chen et al. 2013 Nature 499:295). The GCaMP6f fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). See strain description for more details on development and characteristics of GCaMP6f.

Allele

Symbol: Gt(ROSA)26Sor^{tm95.1(CAG-GCaMP6f)Hze}
Name: targeted mutation 95.1, Hongkui Zeng
MGI accession ID: MGI:5558090
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; No functional change
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: After Cre exposure, EGFP fluorescence is observed following calcium binding (such as neuronal activation).
Molecular note: The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6f coding sequence, a WPRE sequence (to enhance mRNA transcript stability), a bovine growth hormone polyA signal, the GCaMP6f coding sequence (described below), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance mRNA transcript stability), a bovine growth hormone polyA signal, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, and an AttP site. This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. GCaMP6f has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP, and a rat calmodulin DNA fragment.