STOCK Tg(Myh6*/tetO-GCaMP2)1Mik/J

Stock number: 012477
Research areas: Neurobiology Research, Research Tools

Description

This inducible calcium sensor transgene contains a conditional cardiac expressing eGFP-calmodulin fusion protein, GCaMP2. Expression of ccGCaMP2 is regulated by a tetracycline operator (tetO), driven by an enhanced Myh6 promoter which limits GCaMP2 expression in heart. These mice may be useful for measurement of myocyte Ca2⁺ transients in the beating heart of adult and embryonic mice in vivo and in vitro.

Detailed description

This inducible calcium sensor transgene contains a conditional cardiac (cc) expressing eGFP-calmodulin fusion protein, GCaMP2. Mice homozygous for the ccGCaMP2 transgene are viable and fertile, normal in size and do not display any gross physical or behavioral abnormalities. Expression of ccGCaMP2 is regulated by a tetracycline operator (tetO), driven by an enhanced Myh6 promoter which limits GCaMP2 expression in heart. When bred with transgenic mice expressing a tetracycline transactivator (tTA) or reverse transactivator protein, cardiac-specific expression of GCaMP2 can be controlled by withdrawal or administration of tetracycline or a tetracycline analog, doxycycline, in the double mutant offspring. These mice may be useful for measurement of myocyte Ca2⁺ transients in the beating heart of adult and embryonic mice in vivo and in vitro.

Development

This transgene contains a calcium-sensing molecule, GCaMP2, under the transcriptional control of a minimally active mouse alpha myosin heavy chain promoter (Myh6) fused to seven copies of the tetracycline-responsive promoter element (tetO). GCaMP2 consists of, from 5' to 3', a 13-residue peptide of myosin light chain kinase (M13), a circularly permutated eGFP interrupted at residue 145, calmodulin (CaM), and a polyA recognition sequence. Mutations resulting in increased brightness (D180Y and V93I), thermal stability (V163A, and S175G) and GFP dimerization prevention (A206K) were introduced. Three GATA sites and two thyroid-like response elements are ablated in Myh6 to maintain low levels of cardiac-specific expression and decreased background. This conditional cardiac GCaMP2 (ccGCaMP2) construct was injected into fertilized B6D2F2 oocytes and founder mice were bred to FVB/NTac mice to establish a colony. Transgenic offspring were also bred to C57BL/6J prior to arrival at The Jackson Laboratory. Upon arrival mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.

Allele

Symbol: Tg(Myh6*/tetO-GCaMP2)1Mik
Name: transgene insertion 1, Michael I Kotlikoff
MGI accession ID: MGI:3757978
Generation method: Transgenic
Attributes: Inducible; Inserted expressed sequence
Marker symbol: Tg(Myh6*/tetO-GCaMP2)1Mik
Marker name: transgene insertion 1, Michael I Kotlikoff
Chromosome: UN
Strain of origin: (C57BL/6 x DBA/2)F2
Expressed genes: Calm1,calmodulin 1,mouse, laboratory; GFP,Green Fluorescent Protein,; Mylk,myosin, light polypeptide kinase,mouse, laboratory
Site of expression: When bred to mice expressing a tetracycline transactivator (tTA) or reverse transactivator (rtTA) these mice express GFP-calmodulin fusion protein, GCaMP2, in the heart when induced with doxycycline.
Molecular note: This transgene contains a calcium-sensing molecule, GCaMP2, under the transcriptional control of a minimally active mouse alpha myosin heavy chain promoter fused to seven copies of the tetracycline-responsive promoter element (tetO). GCaMP2 consists of circularly permutated eGFP with the molecule interrupted at residue 145 and a 13-residue peptide of myosin light chain kinase (M13) and calmodulin (CaM), placed at the new N and C termini, respectively. An RSET polyHis peptide was introduced N-terminal to the original methionine in GCaMP1. Mutations resulting in increased brightness (D180Y and V93I), thermal stability (V163A, and S175G) and to prevent GFP dimerization (A206K) were introduced. GCaMP2 is 200 times brighter than GCaMP1. Three GATA sites and two thyroid-like response elements are ablated in Myh6 to make the promoter minimally active. When bred with transgenic mice expressing a tetracycline transactivator or reverse transactivator protein, cardiac-specific expression of GCaMP2 can be controlled by withdrawal or administration of a tetracycline analog.