Overview

Also Known As:CHROMus line HCN4-GCaMP8

The fluorescent calcium indicator strain HCN4-GCaMP8 expresses GCaMP8 (a sensitive GCaMP variant with high dynamic range and fast response kinetics) in heart (SA and AV nodes) and nervous system (brain and spinal cord). Upon GCaMP8 binding to calcium, increased EGFP fluorescence is observed.

Donating Investigator

Michael I Kotlikoff, Cornell University

Genetic overview

Genetic Background	Generation

Tg(Hcn4-GCaMP8)B10-3Mik

Allele Type
Transgenic (Reporter)

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Research Applications

Research Tools
Cardiovascular Research
Cell Biology Research
Neurobiology Research

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Base Price

Starting at:

$2,854.50 Domestic price Cryo Recovery

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Details

Detailed Description

HCN4-GCaMP8 transgenic mice express the fluorescent calcium indicator GCaMP8 under control of the Hcn4 locus promoter/enhancer regions within the BAC transgene. HCN4-GCaMP8 transgenic mice from founder line B10-3 (R24:B10:L3) exhibit high sensor expression in heart (SA and AV nodes) and nervous system (brain and spinal cord). Specifically, expression is observed in the developing 13.5 dpc heart and in the brain and spinal cord. In total, transgene expression is consistent with endogenous Hcn4. In the absence of calcium binding, low/baseline EGFP fluorescence is observed. Following calcium binding (such as arteriolar vasodilation), bright EGFP fluorescence is observed at several-fold greater levels than baseline (see GCaMP8 details below).

Hemizygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator reports that breeding hemizygous mice together results in normal numbers of pups. To date (February 2016), it has not been specifically determined if the pups were hemizygous or homozygous genotype.

Calcium is a key molecular signal for cell functions including heart, smooth muscle, vessel, and airway contraction, lung secretion, autonomic neurotransmission and immunocyte function. HCN4-GCaMP8 transgenic mice may be useful to examine calcium signaling in SA and AV nodes both in vivo and in vitro.

The genetically encoded calcium indicator GCaMP8 is a sensitive GCaMP variant with high dynamic range and fast response kinetics (Ohkura et al. 2012 PLoS One 7:e51286). Specifically, the high signal amplitude and low baseline fluorescence of GCaMP8 results in ability to detect single Ca2+ spikes / single neuronal action potentials. GCaMP8 has ~3-fold greater dynamic range than both GCaMP3 and GCaMP6, similar response kinetics to both GCaMP3 and GCaMP6, ~2-fold more rapid decay kinetics than GCaMP3 and lower baseline fluorescence than GCaMP3. 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 bright EGFP fluorescence is observed at ~16-fold greater levels than baseline. In the presence of Ca2+, GCaMP8 has absorbance and emission peaks (~488 nm and ~510 nm, respectively).

This mouse model is available by way of a collaborative effort between Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus) and The Jackson Laboratory.

Development

HCN4-GCaMP8 transgenic mice were designed in the laboratory of Dr. Michael I. Kotlikoff (Cornell University) as part of Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus).
The ~237 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-281H22 was obtained; containing the entire hyperpolarization-activated, cyclic nucleotide-gated K+ 4 locus (Hcn4), as well as ~66 kbp centromeric flanking sequences and ~134 kbp telomeric flanking sequences (including a portion of the Neo1 locus). Using homologous recombination/BAC recombineering, a GCaMP8-pA construct (GCaMP8 followed by an SV40 polyadenylation signal and flanking vector sequence) was inserted into the ATG start site of the BAC Hcn4 gene (replacing the initiation codon of Hcn4 in exon 1). No other loci on the BAC were altered. The resulting ~247 kbp modified BAC (Hcn4^BAC-GCaMP8-pA) was purified and then microinjected into the male pronucleus of FVB x B6(Cg)-Tyr^c-2J/J embryos. Founder animals were bred to C57BL/6J inbred mice for germline transmission. Hcn4^BAC-GCaMP8 founder line B10-3 (R24:B10:L3) was identified with high sensor expression in heart (SA and AV nodes) and nervous system (brain and spinal cord). The donating investigator reports that HCN4-GCaMP8 transgenic mice from this founder line were bred to C57BL/6 for a total of two generations, and then black hemizygous mice were sent to The Jackson Laboratory Repository in 2016. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Of note, the donating investigator reports that, at least once during backcrossing, a hemizygous female was bred to a C57BL/6 inbred male (thus the Y chromosome of the congenic strain is of C57BL/6 origin).

The genetically encoded calcium indicator GCaMP8 is a sensitive GCaMP variant with high dynamic range and fast response kinetics (Ohkura et al. 2012 PLoS One 7:e51286). GCaMP8 is composed of (from N-term to C-term) a polyHis plasmid leader sequence essential for thermal stability (RSET with the ΔH and ΔR2 mutations), a 13 residue peptide of chicken smooth muscle myosin light chain kinase (M13; target peptide for a Ca2+-bound CaM), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with mutations listed below]), a rat calmodulin DNA fragment (CaM; aa 2-148 [with mutations listed below]). The cpEGFP mutations result in improved brightness (D180Y and V93I), thermal stability (V163A and S175G), dimerization prevention (A206K), dynamic range/baseline fluorescence (M153K, T203V, N105Y and E124V), and overall functionality (S205N and I47F). The CaM mutations are for higher calcium affinity (M36L), increased fluorescence change for small calcium transients (N60D) and chromophore stabilization (D78Y).

Expression Data

Expressed Gene	GCaMP, Genetically encoded calcium indicator,
Site of Expression	Transgene expression is consistent with endogenous Hcn4 in the developing heart and in the brain and spinal cord.

Control Suggestions

Noncarrier

Additional Information

Considerations for Choosing Controls

Genetics

Tg(Hcn4-GCaMP8)B10-3Mik

Allele Symbol: Tg(Hcn4-GCaMP8)B10-3Mik

Allele Name	transgene insertion B10-3, Michael I Kotlikoff
Allele Type	Transgenic (Reporter)
Allele Synonym(s)
Gene Symbol and Name	Tg(Hcn4-GCaMP8)B10-3Mik, transgene insertion B10-3, Michael I Kotlikoff
Gene Synonym(s)
Promoter	Hcn4, hyperpolarization-activated, cyclic nucleotide-gated K+ 4, mouse, laboratory
Expressed Gene	GCaMP, Genetically encoded calcium indicator,
Site of Expression	Transgene expression is consistent with endogenous Hcn4 in the developing heart and in the brain and spinal cord.
Strain of Origin	(FVB/N x B6(Cg)-Tyr^c-2J/J)F1
Chromosome	UN
Molecular Note	The ~237 kbp C57BL/6J mouse bacterial artificial chromosome (BAC) RP23-281H22 containing the entire hyperpolarization-activated, cyclic nucleotide-gated K+ 4 locus (Hcn4), as well as ~66 kbp centromeric flanking sequences and ~134 kbp telomeric flanking sequences (including a portion of the Neo1 locus) was modified by inserting the GCaMP8-pA construct (GCaMP8 followed by an SV40 polyadenylation signal and flanking vector sequence) into the ATG start site of the BAC Hcn4 gene (replacing the initiation codon of Hcn4 in exon 1). No other loci on the BAC were altered. The genetically encoded calcium indicator GCaMP8 is a sensitive GCaMP variant with high dynamic range and fast response kinetics. GCaMP8 is composed of (from N-term to C-term) a polyHis plasmid leader sequence essential for thermal stability (RSET with the DeltaH and DeltaR2 mutations), a 13 residue peptide of chicken smooth muscle myosin light chain kinase (M13; target peptide for a Ca2+-bound CaM), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with mutations listed below]), a rat calmodulin DNA fragment (CaM; aa 2-148 [with mutations listed below]). The cpEGFP mutations result in improved brightness (D180Y and V93I), thermal stability (V163A and S175G), dimerization prevention (A206K), dynamic range/baseline fluorescence (M153K, T203V, N105Y and E124V), and overall functionality (S205N and I47F). The CaM mutations are for higher calcium affinity (M36L), increased fluorescence change for small calcium transients (N60D) and chromophore stabilization (D78Y).

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Research Tools
- Genetics Research
  - Tissue/Cell Markers
  - Tissue/Cell Markers: endothelial cells
- Fluorescent Proteins
- Cell Biology Research
- Cardiovascular Research
Cardiovascular Research
- Heart Abnormalities
- Other
Cell Biology Research
- Channel and Transporter Defects
  - calcium
Neurobiology Research
- Channel and Transporter Defects
  - calcium
- Optogenetic and Chemogenetic tools
  - Calcium sensor expressing strains

Mammalian Phenotype Terms by Genotype

References

Additional - Tg(Hcn4-GCaMP8)B10-3Mik related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Standard PCR:Tg(Hcn4-GCaMP8)B10-3Mik
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, hemizygous mice may be bred to wildtype (noncarrier) mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). The donating investigator reports that breeding hemizygous mice together results in normal numbers of pups. To date (February 2016), it has not been specifically determined if the pups were hemizygous or homozygous genotype.
- Additional Breeding and Husbandry Support
Citation
When using the CHROMus line HCN4-GCaMP8 mouse strain in a publication, please cite the originating article(s) and include JAX stock #028344 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Pricing & Availability

Cryo Recovery

Domestic
International

Live Mouse

Age

Genotype

Price

weeks

Cryorecovery - Pricing

Service/Product	Description	Price
	Hemizygous or non-carrier for Tg(Hcn4-GCaMP8)B10-3Mik

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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.

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Also Known As:CHROMus line HCN4-GCaMP8

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Additional Information

Tg(Hcn4-GCaMP8)B10-3Mik

Allele Symbol: Tg(Hcn4-GCaMP8)B10-3Mik

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

References

Additional - Tg(Hcn4-GCaMP8)B10-3Mik related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Pricing

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms Of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability