B6.Cg-Igs7^{tm143.1(tetO-RCaMP1.07)Hze}/J

Stock number: 030217
Product name: TITL-R-CaMP1.07-D (or Ai143D)
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

TITL-R-CaMP1.07-D (Ai143D) mice are a Cre/Tet-dependent, fluorescent calcium-indicator tool strain, created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Following Cre-mediated removal of the STOP cassette, they may be used to generate Tet-Off/Tet-On mutant animals with conditional (inducible/reversible) expression of R-CaMP1.07 (a red fluorescent calcium-sensing molecule that functions at cellular calcium levels).

Detailed description

TITL-R-CaMP1.07-D (Ai143D) mice are a Cre/Tet-dependent, fluorescent calcium-indicator tool strain, created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). TITL-R-CaMP1.07-D mice harbor the TIGRE-Ins-TRE-LSL-R-CaMP1.07 conditional allele, designed with a modified Tet response element (TRE or tetO) and loxP-flanked STOP cassette upstream of the fluorescent calcium-indicator R-CaMP1.07 protein (see detailed description below). When bred with other mice expressing Cre recombinase, tetracycline-controlled transactivator protein (tTA) and/or reverse tetracycline-controlled transactivator protein (rtTA), R-CaMP1.07 is expressed in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap, and R-CaMP1.07 expression can be regulated with tetracycline or its analog doxycycline (dox). Once R-CaMP1.07 is expressed, low/baseline RFP fluorescence is observed in the absence of calcium binding. Following calcium binding (such as neuronal activation), a conformational change occurs that results in brighter RFP fluorescence (see R-CaMP1.07 details below).

Specifically, the donating investigator reports in the absence of Cre and tTA, mice heterozygous for TITL-R-aMP1.07-D exhibit no RFP fluorescence, and are viable and fertile with no reported gross physical or behavioral abnormalities. To date (March 2017), it has not been attempted to make this strain homozygous.
When TITL-R-CaMP1.07-D are bred to tTA-driver and Cre-driver lines with overlapping expression in all cortical layers, the resulting triple transgenic animals (R-CaMP1.07+/Cre+/tTA+) have good calcium-dependent responses under in vivo 2-photon imaging. Multiple action potentials evoke much bigger fluorescence changes (compared to baseline fluorescence). When using TITL-R-CaMP1.07-D with driver lines Camk2a-tTA, Rasgrf2-2A-dCre and Nr5a1-Cre (see Stock Nos. 007004, 022864 and 006364, respectively), the donating investigator reports calcium reporter function is very efficient, however fluorescence signal may appear punctate.

R-CaMP1.07 expression/function in tissues other than brain has not yet been evaluated by the donating investigator (March 2017).

The genetically encoded calcium indicator R-CaMP1.07 is a red calcium-sensing molecule that functions at cellular calcium levels (Ohkura et al. 2012 PLoS One 7:e39933). R-CaMP1.07 is composed of a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated red fluorescent protein (mApple; with amino acid substitutions designed to increase Ca2+-dependent fluorescent change), a rat calmodulin sequence and a self-cleaving peptide (F2A; to increase the Ca2+-dependent fluorescent change and facilitate export from the nucleus). In the absence of calcium binding, low/baseline RFP fluorescence is expected. Upon calcium binding, a conformational change occurs that results in bright RFP fluorescence at ~28-fold greater levels than baseline. In the presence of Ca2+, R-CaMP1.07 has absorbance and emission peaks (~562 nm and ~584 nm, respectively) that are ~80nm red-shifted compared to most GFP-based calcium indicators. Of note, R-CaMP1.07 may not be appropriate for imaging of neuronal activity in combination with optogenetic channel activation (e.g., channelrhodopsin 2 [ChR2]). The red fluorescent Ca2+ indicator R-GECO1 (from which R-CaMP1.07 was derived) is reported to exhibit some blue light-induced photoactivation, which may cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. If using red fluorescent Ca2+ indicators along with ChR2, the researcher may try to avoid artifacts by using an appropriately low intensity of blue light for ChR2 activation.

Development

The TITL-R-CaMP1.07-D (Ai143D) allele has a Tet response element, floxed-STOP cassette and R-CaMP1.07 sequence inserted into the T1 TIGRE locus (an intergenic region on chromosome 9 [between AB124611 and Carm1] determined to have low basal transcriptional activity and allow high Cre and/or Tet inducibility).

Specifically, the pTRE-LSL-R-CaMP1.07 replacement vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a modified Tet response element/promoter (TRE/tetO), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the R-CaMP1.07 sequence (details below), a WPRE (to enhance mRNA stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site.
(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3'hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the pTRE-LSL-R-CaMP1.07 replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with C57BL/6J-congenic PhiC31 mice (see Stock No. 007743) to remove the AttB/AttP-flanked PGK-hygro-SV40polyA and replace it with the recombined AttB/AttP site (AttL). The TITL-R-CaMP1.07-D allele is therefore TIGRE-frt3-Ins-TRE-LSL-R-CaMP1.07-WPRE-bGHpA-Ins-AttL. The resulting TITL-R-CaMP1.07-D mice were bred several generations to C57BL/6J (the PhiC31 gene was removed). In 2017, generation N4 males (black coat color) were sent to The Jackson Laboratory and their sperm was cryopreserved (48 SNP panel showed all markers were C57BL/6J allele-type). To establish our live colony, frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
The donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J male (thus the Y chromosome is of C57BL/6J origin).

The genetically encoded calcium indicator R-CaMP1.07 is a red calcium-sensing molecule that functions at cellular calcium levels (Ohkura et al. 2012 PLoS One 7:e39933), and is a modified version of R-CaMP1.01 (which was created by insertion of random mutations into R-GECO1). R-CaMP1.07 is composed (from N-term to C-term) of a 35 aa polyHis plasmid leader sequence (RSET; essential for thermal stability), a 13 aa chicken smooth muscle myosin light chain kinase (M13; target peptide for a Ca2+-bound CaM), a circularly permutated red fluorescent protein (mApple; aa 146-231 followed by aa 1-145 [with mutations described below]), a rat calmodulin DNA fragment (CaM; aa 2-148), a 15 aa linker and a self-cleaving peptide (F2A). Compared to R-GECO1, the mApple mutations K47V/T49V result in increased Ca2+-dependent fluorescent change. The C-terminal F2A also increases the Ca2+-dependent fluorescent change, as well as R-CaMP1.07 nuclear export. For the TITL-R-CaMP1.07-D allele, the plasmid encoding R-CaMP1.07 was supplied by Dr. Fritjof Helmchen (University of Zurich).

The TRE promoter used here is Tet-responsive P_tight; containing a modified Tet response element (TRE_mod) composed of seven direct 36 bp repeats (each containing the 19 bp tet operator sequence [tetO]). TRE_mod is just upstream of a minimal human cytomegalovirus promoter (P_minCMVΔ), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_tight is silent in the absence of tTA or rtTA binding to tetO.

Allele

Symbol: Igs7^{tm143.1(tetO-RCaMP1.07)Hze}
Name: targeted mutation 143.1, Hongkui Zeng
MGI accession ID: MGI:5903975
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible
Synonyms: Ai143D; TITL-R-CaMP1.07-D
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: RCaMP,Red genetically encoded calcium indicator,
Site of expression: When bred with other mice expressing Cre recombinase, tetracycline-controlled transactivator protein (tTA) and/or reverse tetracycline-controlled transactivator protein (rtTA), R-CaMP1.07 is expressed in cells/tissues where the expression patterns of the individual promoters driving Cre and tTA/rtTA overlap, and R-CaMP1.07 expression can be regulated with tetracycline or its analog doxycycline (dox).
Molecular note: The replacement vector is designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element, a modified Tet response element/promoter, a floxed-STOP cassette, the R-CaMP1.07 sequence (details below), a WPRE, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. Embryonic stem cells previously targeted with FRT3::AttB::PGK-neoR-polyA::FRT5::splice acceptor::3'hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the pTRE-LSL-R-CaMP1.07 replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME). PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL). R-CaMP1.07 is a red calcium-sensing molecule that functions at cellular calcium levels and is a modified version of R-CaMP1.01 (which was created by insertion of random mutations into R-GECO1). R-CaMP1.07 is composed (from N-term to C-term) of a 35 aa polyHis plasmid leader sequence, a 13 aa chicken smooth muscle myosin light chain kinase, a circularly permutated red fluorescent protein (mApple; aa 146-231 followed by aa 1-145), a rat calmodulin DNA fragment, a 15 aa linker and a self-cleaving peptide (F2A).