C57BL/6-Igs2^{tm6(CAG-GFP*/TPTE2*)Mzlin}/J

Stock number: 041169
Product name: ASAP5-Kv
Research areas: Neurobiology Research, Research Tools

Description

ASAP5-Kv mice are a Cre-inducible, voltage-sensitive, fluorescent line, created by targeted insertion at the Igs2 locus (Hipp11 or H11; an intergenic region on chromosome 11). Exposure to Cre recombinase removes a STOP cassette, resulting in expression of ASAP5 (green fluorescent voltage indicator with high sensitivity) in cre-expressing cells/tissues. These mice may be useful for in vivo imaging of voltage dynamics.

Detailed description

ASAP5-Kv mice are a Cre-inducible, voltage-sensitive, fluorescent line, created by targeted insertion of a CAG-LSL-ASAP5-Kv cassette into the Igs2 locus (Hipp11 or H11; an intergenic region on chromosome 11). Accelerated sensor of action potential (ASAP)-type sensors have a circularly permutated fluorescent protein linked to the transmembrane helix of a voltage-sensing domain (VSD). Voltage-induced movement of a positively charged helix in VSD is thought to perturb the protonation state of the circularly permuted fluorescent protein, resulting in changes in fluorescence emission. ASAP5 is a higher-sensitivity variant of the green fluorescent voltage indicator ASAP3, that has amino acid replacements D145A, R149K, I412V, F413I and Q414R in the VSD sequences (GgVSD) from the Gallus gallus voltage-sensitive phosphatase TPTE2 locus (Hao et al., 2024 Neuron).

Heterozygous and homozygous ASAP5-Kv mice are viable and fertile with no overt phenotype. When bred to mice expressing Cre recombinase, resulting progeny will have the loxP-flanked STOP cassette removed, leading to expression of ASAP5 (green fluorescent voltage indicator with high sensitivity) in cre-expressing cells/tissues. These mice may be useful for in vivo imaging of voltage dynamics in neuronal populations.

Development

The ASAP5-Kv allele was generated by insertion of a CAG-LSL-ASAP5-Kv expression cassette, flanked by attB sites, into the Igs2 locus (Hipp11 or H11; an intergenic region on chromosome 11 [cytoband A1 at ~3cM between the Eif4enif1 and Drg1 loci] reported to support high-level global gene expression in conjunction with the CAG (CAGGS) promoter and a higher rate of mitotic/interchromosomal recombination compared to Gt(ROSA)26Sor). Specific details below.

The CAG-LSL-ASAP5-Kv target cassette includes (from 5' to 3') an initial attB site, a cytomegalovirus enhancer/chicken beta-actin core promoter (CAG), a loxP site, a 4xSV40polyA cassette, a second loxP site, a fast genetically encoded voltage indicator (ASAP5), which is composed of a circularly permuted GFP (cpGFP) inserted in an extracellular loop of a voltage-sensing domain (VSD), fused to a 65-amino-acid cytoplasmic segment of the Kv2.1 potassium channel, followed by an LKGSSGSSGSSTR linker, a second SV40polyA signal and a second attB site (CAG-loxP–SV40polyA–loxP-ASAP5-Kv-SV40polyA). The H11-ASAP5-Kv donor plasmid was injected into B6H11 zygotes along with a PhiC31 integrase expression plasmid, followed by genotyping for insertion at the H11 attP site. The resulting chimeric mice were bred to C57BL/6 mice to confirm germline transmission and maintained on a C57BL/6 background by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

ASAP5 is a higher-sensitivity variant of the green fluorescent voltage indicator ASAP3, that has a circularly permutated GFP linked to the transmembrane helix of a voltage-sensing domain (VSD), and contains the additional amino acid replacements D145A, R149K, I412V, F413I and Q414R (Hao et al., 2024 Neuron).

Allele

Symbol: Igs2^{tm6(CAG-GFP*/TPTE2*)Mzlin}
Name: targeted mutation 6, MIchael Z Lin
MGI accession ID: MGI:8394068
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Igs2
Marker name: intergenic site 2
Marker synonyms: H11; Hipp11; Iis2
Chromosome: 11
Strain of origin: C57BL/6
Molecular note: The allele is created using site-specific PhiC31 integrase-mediated transgenesis to insert an initial attB site cytomegalovirus enhancer/chicken beta-actin core promoter (CAG), a loxP site, a 4xSV40polyA cassette, a second loxP site, a fast genetically encoded voltage indicator (ASAP5), which is composed of a circularly permuted GFP (cpGFP) inserted in an extracellular loop of a voltage-sensing domain (VSD), fused to a 65-amino-acid cytoplasmic segment of the Kv2.1 potassium channel, followed by an LKGSSGSSGSSTR linker, a second SV40polyA signal and a second attB site (CAG-loxP–SV40polyA–loxP-ASAP5-Kv-SV40polyA). The H11-ASAP5-Kv donor plasmid was injected into B6H11 zygotes along with a PhiC31 integrase expression plasmid.