B6J.Cg-Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}/MwarJ

Stock number: 028866
Product name: Ai96(RCL-GCaMP6s) (C57BL/6J) or Ai96 (C57BL/6J)
Research areas: Neurobiology Research, Research Tools

Description

Ai96(RCL-GCaMP6s) (also called Ai96) mice are a Cre-dependent, fluorescent, calcium-indicator tool strain. Ai96 has a floxed-STOP cassette preventing transcription of the GCaMP6 slow variant calcium indicator (GCaMP6s; an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics). After Cre exposure, increased EGFP fluorescence is observed following calcium binding (such as neuronal activation).

Detailed description

Stock No. 028866 is the C57BL/6J-congenic Ai96 mouse line (Ai96 (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 Ai96 mice on a mixed genetic background (Stock No. 024106). 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.

Ai96(RCL-GCaMP6s) mice (also called Ai96) harbor the Rosa-CAG-LSL-GCaMP6s conditional allele, designed with a floxed-STOP cassette upstream of the GCaMP6 slow variant calcium indicator (GCaMP6s; see detailed description below). Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of GCaMP6s is prevented by STOP cassettes. After exposure to Cre recombinase, EGFP fluorescence is observed following calcium binding (such as neuronal activation).

Specifically, the donating investigator reports Ai96 mice have no reported levels of EGFP fluorescence prior to exposure to Cre recombinase. When Ai96 mice are bred with a Cre-driver line to create double transgenic animals (GCaMP6s+/Cre+), cells expressing Cre exhibit low EGFP fluorescence in the absence of calcium binding. Following calcium binding (such as neuronal activation), increased EGFP fluorescence is observed in these cells.

The donating investigator also reports that following neuronal activation, the fluorescent intensity observed in double transgenic Ai96 mice (GCaMP6s+/Cre+) is less than that of the double transgenic Ai95 system using GCaMP6 fast variant calcium indicator (GCaMP6f+/Cre+ ; see Stock No. 024105). Although both systems utilize Cre-dependent GCaMP6/EGFP fusion genes, they differ in the kinetics and sensitivity of their GCaMP6 variants.

Heterozygous Ai96 mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator has not attempted to generate homozygous Ai96 mice to date (March 2014).

For characterization information of the Ai96 allele, see images at the Allen Institute for Brain Science website (Ai96(RCL-GCaMP6s) images).

The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. The GCaMP6s fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). Compared to GCaMP6f, the slower kinetics render GCaMP6s more sensitive and slower to decay. GCaMP6s has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (with several amino acid substitutions designed to optimize dynamic range, baseline fluorescence and sensitivity), and a rat calmodulin sequence modified to increase the fluorescence change for small calcium transients. Other modifications in the cpEGFP-to-CaM linker also improve sensor function. 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.

Development

The Ai96(RCL-GCaMP6s) allele (also called Ai96) was designed by Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a floxed-STOP cassette upstream of the GCaMP6 slow variant calcium indicator (GCaMP6s), all inserted into the Gt(ROSA)26Sor locus. Specific details below.

The Rosa-pCAG-LSL-GCaMP6s-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 GCaMP6s coding sequence, a WPRE sequence (to enhance mRNA transcript stability), a bovine growth hormone polyA signal, the GCaMP6s 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. The resulting Ai96 mice were bred to C57BL/6J wildtype mice for at least one additional generation. In 2014, Ai96 heterozygous males on a mixed genetic background (estimated 75% 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. 024106, 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. 024106 animals (total backcross generation N3 onto C57BL/6J). They further backcrossed them to C57BL/6J inbred mice for four additional generations. The resulting C57BL/6J-congenic strain, called Ai96(RCL-GCaMP6s) (C57BL/6J) or Ai96 (C57BL/6J), were sent to The Jackson Laboratory Repository in 2016 as Stock No. 028866. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664).
Of note, Dr. Warden's lab reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slow response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. The GCaMP6s fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). GCaMP6s 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, as well as K166H mutation for increased sensitivity/slower kinetics]), 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. The T302L and R303P amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function.

Allele

Symbol: Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}
Name: targeted mutation 96, Hongkui Zeng
MGI accession ID: MGI:5558094
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inserted expressed sequence
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 exposure to Cre recombinase, 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 GCaMP6s coding sequence, a WPRE sequence (to enhance mRNA transcript stability), a bovine growth hormone polyA signal, the GCaMP6s 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. GCaMP6s has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP, and a rat calmodulin DNA fragment to increase the fluorescence change for small calcium transients.