Fluorescent protein transgenic mice for the study of Ca²⁺ and redox signaling

Summary

Many unanswered questions of physiology and medicine require in vivo studies of cellular processes in murine models. These processes commonly depend on intracellular Ca²⁺ and redox alterations. Fluorescent dyes have succeeded in real-time intracellular monitoring of Ca²⁺, redox and the different Reactive Oxygen Species (ROS) in single cells, but have seldomly been applied in vivo. The advance in Fluorescent Protein (FP) technology has created alternative tools for the same task, which can be delivered with viruses or genomic integration strategies into mice. With the availability of several color options for both Ca²⁺ and redox reporting FP, multiparameter measurements have also become feasible: measuring different species, and the same parameter at different locations using organelle-specific targeting sequences at the same time. We, here, focus on mice with genomic integration of Ca²⁺ and redox reporters, provide a list of the available models and summarize the strategies of their generation and utilization. We also describe a novel Calcium DoubleSpy mouse model that conditionally expresses both RCaMP in the cytoplasm and GEM-GECO1 in the mitochondrial matrix, allowing the study of mitochondrial Ca²⁺ related physiology and pathogenesis simultaneously in two distinct intracellular compartments.

Need for monitoring intracellular Ca²⁺ and redox/ROS in single cells in mice

The concentration of Ca²⁺ ([Ca²⁺]) is maintained in the cytoplasm ([Ca²⁺]_c) and within the lumen of some intracellular organelles like the mitochondrial matrix [Ca2+] ([Ca²⁺]_m) at ~100 nM. This is 4 orders of magnitude lower than the [Ca²⁺] in the extracellular solutions and endoplasmic reticulum. Transient elevations of [Ca²⁺]_c and [Ca²⁺]_m control many essential tissue functions including neurosecretion, cardiac and skeletal muscle contractions and metabolism, but sustained increases in the “low [Ca²⁺]” intracellular zones can cause cell injury and death [1, 2].

The flow of electrons underpins reduction and oxidation “redox” reactions that are necessary for organisms to extract and direct energy. Cells invest energy in maintaining the redox balance in various subcellular compartments with the general intracellular environment kept at a significantly reduced redox potential relative to the extracellular environment by interlinked redox couples such as NAD(H), NADP(H) FAD(H2) and glutathione (GSH:GSSG). This environment confers protection against oxidation to proteins, nucleic acids and lipids which are themselves highly reduced polymers. Redox reactions commonly produce the various forms of ROS, such as superoxide anion (O₂^•−), and secondary products, hydrogen peroxide (H₂O₂), and peroxynitrite (ONOO^•−). Transient elevations of ROS are central to physiological signaling, whereas prolonged and excessive ROS, like Ca²⁺ cause cell injury and death [3].

ROS and Ca²⁺ elevations share similarity not only as fundamental signaling entities and cell injury inducers, but also act synergistically on specific targets and controlling generation and elimination of each other. They directly interact with and alter conformation and function of a range of proteins as well as lipid membranes [2, 4]. Imbalance in any of these ions can be harmful or fatal for the cell [4]. In recent decades, mitochondria have emerged as central hubs in these signaling systems and as regulators of both Ca²⁺ and ROS homeostasis [4]. The principal sites in the production of ROS are the mitochondrial respiratory complexes I and III. The synergism between Ca²⁺ and ROS centers on the mitochondrial permeability transition pore (PTP). Sustained PTP opening stimulated by mitochondrial Ca²⁺ overload, ROS excess and other factors presents a trigger for cell death, while transient openings appear to constitute a signaling mechanism.

In isolated cells, the spatiotemporal organization of Ca²⁺/ROS signaling and Ca²⁺/ROS toxicity mechanisms and their various compartments have been investigated. However, the information gained from these studies might not be directly applicable to the in vivo situation where a higher level of complexity likely exists based on intercellular interactions that often involve many different cell types. Indeed, Ca²⁺ imaging studies performed in specific tissues revealed the coordination and shaping of Ca²⁺ signals across many cells [5–7]. Another level of complexity in vivo is created by the interactions among the different tissues. These observations highlight the need of recording intracellular Ca²⁺ and ROS in various tissues in vivo to address these complexities.

Monitoring Ca²⁺ and ROS by fluorophores

Information about specific aspects of cell and tissue function were first obtained by measuring their output into the extracellular space, and by testing various fractions prepared from broken-up cells and tissues. Radioisotopes, colorimetric probes and chemiluminescence were the pioneers as tracers within the cells of living organisms. However, the measurements of intracellular [Ca²⁺] and ROS became feasible in a variety of cell types and in even some tissues with the development of fluorescent indicators. For a historic perspective and critical assessment of fluorescent dyes versus the preceding technologies see [8] by the late Roger Y. Tsien who is credited as the main driver of the fluorescent dye and fluorescent protein technologies.

Ca²⁺ sensing fluorescent dyes are derivatives of the selective Ca²⁺-chelators, EGTA and BAPTA. The changes in the excitation and/or emission properties of the dyes result from the conformational change caused by Ca²⁺ binding to the carboxyl groups, which is transmitted to the chromophore introduced to EGTA. The synthesis of the acetoxymethyl (AM) ester of these indicators made their loading into almost every cell type simple. This strategy was originally planned so that the cleavage products of the -AM form would be trapped in the cytoplasm. However, modifications of the loading conditions allowed trapping of a large fraction of the dyes within intracellular organelles including the mitochondrial matrix and endoplasmic reticulum lumen, where esterases can also free the Ca²⁺-sensitive and membrane-impermeant fluorescent dye from the AM ester. The Ca²⁺-sensitive fluorescent dye technology has benefited from 40+ years of development and provides both intensiometric and ratiometric options, in addition to a range of excitation and emission wavelengths. The commonly used current options are descendants of fura2, fluo3 and rhod2. Notably, adapting the strategy of the Ca²⁺ indicators to other ions gave rise to fluorescent indicators for intracellular pH and [Na⁺]. A significant limitation of these fluorescent dye-based probes is that their localization is rather non-specifically controlled via empirical adjustment of the physicochemical properties of the dye (e.g. net charge) and the loading conditions.

For decades, measurements of ROS in cells have relied upon a small group of intensiometric fluorescent indicators based upon reduced ethidium, fluorescein or rhodamine. Dihydroethidium and MitoSOX, a charged derivative that partitions to mitochondria, show increased fluorescence upon oxidation by O₂^•−. However, non-specific oxidation generates a similarly bright product with an overlapping spectrum [9]. To confirm O₂^•− measurement, spectral analysis or purification/HPLC analysis is required. Dichlorodihydrofluorescein diacetate (DCFH-DA) is a reduced, cell permeable ester that is hydrolyzed in the intracellular environment to dihydroxy-DCFH. Because the chemistry occurs in the cytosol, this dye is commonly used for detecting intracellular H₂O₂ [10]. However, DCFH-DA and derivatives suffer from multiple potential problems including: a lack of direct/specific reactivity with H₂O₂, the possibility that the fluorescent product DCF may generate O₂^•− /H₂O₂ through reaction with O₂, and the fact that transition metals/cytochrome c can catalyze DCFH oxidation [11]. Dihydrorhodamine (DHR), commonly used for ONOO^•− detection, relies upon oxidative conversion of DHR to its fluorescent two-electron oxidized product, rhodamine. However, oxidation of DHR involves an intermediate DHR radical vulnerable to reduction by thiols/ascorbate. This renders DHR useful only as a nonspecific indicator of intracellular ONOO^•−, HOCl or other one-electron oxidants [12]. Thus, all these probes suffer from problems with specificity and artifacts and are non-reversible.

In trying to circumvent some of the limitations cited above, a new family of fluorophores protected by boronate groups with multiple fluorescence spectra (blue, green & red) and cytosolic/mitochondrial partitioning were developed. The fluorescence yield of the parent fluorophore increased when exposure to µM H₂O₂ removed the protective group (Miller et al., 2005). Quinone chemistry was also used to synthesize H₂O₂ probes in the NIR range [13]. A family of boronate probes directly responsive to ONOO•− has also been generated [14]. Notably, Miller and colleagues did not test for ONOO•− sensitivity and Zielonka et al. reported H₂O₂ cross-reactivity, raising potential specificity issues. Using redox-sensitive proteins as a model, a fluorescein-based loadable dye equipped with a reversible thiol pair was generated [15]. Similarly, probes based on the selenoprotein core of GPx which catalyzes reactions with GSH and hydroperoxides/thiols have been developed to integrate between specific redox pairs including ONOO- /thiols, HOCl/H₂S, HOCl/GSH, HOBr/H₂S probe [16–19]. More exotic organotellurium dyes were also created which feature enhanced GPx-activity that insert between ONOO- /Thiols, available with NIR spectrum and some organelle-specific (mitochondrial) targeting (Yu et al., 2013). Of great biological interest is detection of O₂^•−. Recently, a new probe that inserts between superoxide and the thiol pool has been demonstrated [20]. This probe, also based upon organoselenide/telluride catalysis, constitutes a reversible probe to follow superoxide dynamics.

In summary, it seems clear that the newer redox sensitive fluorophores provide several advantages, including the fact that they are reversible, they seem more specific and many are available in multiple or more useful fluorescence spectra. However, a drawback of these new probes is that they remain relatively untested in terms of time and number of studies. Furthermore, how a range of relatively novel chemistries react with the intracellular environment of diverse cell types should be considered when experiments are planned.

Fluorescent Protein Technology

Green fluorescent protein (GFP), isolated from the jellyfish, Aequorea victoria, was a breakthrough finding. It is now routinely used as a fluorescent label that can be incorporated into proteins by genetic fusion. However, to take GFP from a curiosity of marine biology to a widely used laboratory tool, several development steps were taken. These included including mutations to enable folding/stability at 37°C, and shifting of the excitation spectrum to enhance brightness and remove the tendency of GFP to dimerize. Modern GFPs can be expressed in situ by gene transfer into cells and localized to subcellular compartments with appropriate targeting signals, thereby allowing imaging of a range of biological events. The color palette has expanded to blue, cyan, and yellow variants of GFP, and a whole palette of red-shifted colors developed from the coral-derived dsRed, which, also required significant development of mutations to produce a more useful monomer (mRFP) and spectrally shifted variants. Multiple colors of FPs can be used at the same time to facilitate multiparameter imaging measurements. The FPs offer an opportunity for photobleaching recovery experiments, and photoactivatable and photoswitchable FP variants have also been created. Fusion of FPs with proteins of interest makes possible the visualization of the dynamics of particular cellular proteins. Fusion of FPs with membrane targeting sequences or lipid binding motifs allows the study of specific organellar or lipid dynamics. FPs have been exploited for a broad range of “intracellular spying” purposes but herein, we will focus on the FP strategies that allow Ca²⁺ and redox monitoring.

Fluorescent protein based Ca²⁺ and redox spying

The fluorescent Genetically Encoded Ca²⁺ Indicators (GECI) include a Ca²⁺ sensing domain, calmodulin or troponin C, which have 4 Ca²⁺ binding EF hands, fused to 1 or 2 FP. The first GECIs were the Cameleons which contain two fused FPs, a Förster resonance energy transfer (FRET) pair, and produce an emission ratio signal reflecting Ca²⁺-dependent changes in the efficiency of the FRET. A single FP-based ratiometric and pH-insensitive probe is GEM-GECO1 that exhibits a shift in the emission spectra when bound to Ca²⁺. However, most single FP-based GECI are intensiometric, meaning, they change fluorescence intensity when bound to Ca²⁺. Pericam and GCaMP exploited a circularly permuted FP topology. A recent and broadly used family is the GCaMP6 series. Color variants include blue, cyan, yellow, and red Ca²⁺ indicators. Red fluorescent variants are R-GECO1, RCaMP, and the newer jRCaMP and jRGECO1. Many of the newer sensors (jGCaMP, jRGECO and jRCaMP) are generally very high affinity since these GECI are primarily used for catching small [Ca²⁺] fluctuations associated with action potentials in neurons. Red GECI are preferable to green ones due to the lower autofluorescence, and deeper tissue penetration associated with longer wavelength excitation.

A relatively new line of optogenetic Ca²⁺ reporters have been developed to report on [Ca²⁺] signaling activity in an integrative post hoc manner so that active cells in a population could be quantitatively identified based on Ca²⁺-dependent irreversible green→red photoconversion of the FP probe. The prototype FP was dubbed CaMPARI1 (calcium-modulated photoactivatable ratiometric integrator) [21] and later its improved version CaMPARI2 [22]. For the latter, a monoclonal antibody has also been developed against the photoconverted red form, allowing immuno-visualization in fixed cells/tissue. CAMPAR1/2 have so far been primarily used for marking active neuronal populations; however, it is expected that their use as a new generation of Ca²⁺ reporters will likely broaden.

The development of fluorescent Genetically Encoded Redox Indicators (GERIs), that allow real-time visualization of the oxidation state of the FP, stimulated a new era in redox biology. They feature a common mechanism whereby the fluorescence property of the FP is modulated by structural changes induced by the reversible oxidation/reduction of a disulfide bridge between the thiol sidechains of two surface-exposed cysteine residues. The first such probe was rxYFP [23] followed with roGFP1 and roGFP2 [24, 25]. Their disulfide/thiol pair equilibrates with the thiol pool dominated by GSH:GSSG. roGFP1 and roGFP2 are monomeric, excitation-ratiometric probes, which do not exhibit major pH sensitivities common to YFP-derived probes [25]. Further modification and improvement of these probes increased the response rate (roGFP1Rx) [26] and adapted the redox midpoint to more oxidizing environments [27]. An important development of roGFP1/2 was made when human glutaredoxin 1 or 2 (Grx1/Grx2) was added to catalyze the reaction between the GSH and the roGFP thiol pair dramatically increasing its selectivity and response rate [28]. Grx1roGFP2 allows imaging of redox dynamics at sub-second temporal resolution [29].

The measurement of specific ROS with GERIs has largely focused upon the relatively long-lived H₂O₂ molecule produced either directly, or via dismutation of O₂^•. To do this, a more specialized thiol pair derived from the E.coli OxyR transcription factor is used, containing a cysteine residue buried inside a hydrophobic pocket that selects for H₂O₂ and forms a disulfide bond that modulates the fluorescence of cpYFP to create the probe HyPer [30]. This probe detects H₂O₂ at physiologically relevant concentrations with fast temporal resolution [31] but retains the pH sensitivity problems of rxYFP. Systematic developments of the concept have improved the brightness, response and pH sensitivity of HyPer [32]. H₂O₂ insensitive mutants based upon the C121S substitution in the OxyR domain often termed, “SypHer” are recommended to control for pH-induced artifacts when using HyPer [33, 34]. In addition, a red-shifted derivative, HyPerRed was created, which allows combination of HyPerRed with a green fluorescent reporter in multiparameter measurements [35]. Further developments by other groups have focused upon the discovery in various microbial species of natural protein sensors of H₂O₂. The most developed of these is the Yeast Orp1 domain, which has been used to confer H₂O₂ specificity to existing probes including roGFP [36], and a pair of FRET probes PerFRET & OxyFRET that offer pH resistance and interesting spectral opportunities [34].

At this point in time, GERI probes offer the investigator opportunities to probe dynamic changes in the intracellular redox environment which is dominated by the GSH thiol pool, and to monitor specifically H₂O₂. While related species such as O₂^•− and ONOO^•− may be of great interest, development of a GERI for these species awaits a validated protein sensor that might be engineered into a functional probe.

Reporter gene genomic integration and construction strategies in mice

There are two principal considerations for the genomic integration of reporter FP constructs. When the reporter protein is used as a tag on a functional endogenous protein, the physiologically most inert option is to swap the native allele with the tagged fusion protein allele via knock-in (or gene editing). In this case, the construct integrates into the corresponding native/endogenous gene locus. However, the reporter is often intended to spy on subcellular compartment(s) independently from an endogenous protein. In such cases, genomic integration may happen via random insertion, as a transgenic construct (Tg) or as an artificial chromosome (bacterial, BAC or yeast, YAC) (see JAX mice with insertion sites indicated as ‘random’ or unknown’ in Table 1). Random genomic integration is relatively simple strategy, but it increases the risk of off-target effects. Instead of random integration, knock-in strategies to so called safe harbor loci have been developed. The most commonly used safe harbor loci are ROSA26 (on chromosome 6) [37] and the intergene region TIGRE/Igs7 (on chromosome 9) [38, 39] (see corresponding mice in Table 1). Other intergene (noncoding) chromosomal regions have also been used, for example, the transposon insertion site downstream Polr2a (m chromosome 11) [40], Chr13 between 5710045 and 5710046 [41] and, Chr1 position 174,611,546 [42] (Table 1).

Table 1a.

Reporter mice available for [Ca²⁺]_c

Spying on	Reporter	Ex/Em (nm)	Insertion site	Constitutive/Conditional (cell type)	Source	Deposited Jackson #
[Ca2+]c signal history	CaMPARI2	PC @405 G: 502/516 R: 562/577	Rosa26 Gt(ROSA)26Sortm1(CAG-Eos*/Calm*)	Conditional (STOPfl/fl)/ whole body (CAG)	UP	34240
[Ca2+]c	YCX2.60	Em ratio -: 432/480 Ca:432/528	IGs7/TIGRE Igs7tm92.1(tetO-FP)	2x Conditional (STOPfl/fl, TetO)	[38]	26262
			Random (BAC) Cx40BAC-FP	Constitutive, Endothel, atr. Myocytes (Cx40)	[63]	30333
	GCaMP2	485/510	Random (BAC) Acta2BAC-FP	Constitutive, Sm muscle (Acta2)		25405
			Random (BAC) Cx40BAC-FP-IRES-FP (2x FP)	Constitutive, Endothel, atr. Myocytes (Cx40)	[64]	25404
			Unknown pCAGGS-FPfl/fl	Conditionally removable (Cre)/ whole body (CAG)		25619
			Unknown Tg(tetO-FP)	Conditional (TetO)	[65]	17755
			Unknown Tg(Myh6*/tetO-FP)	Conditional (TetO)/ heart (Myh6)	[66]	12477
	GCaMP2.2c	485/510	Unknown Tg(Thy1-FP)	Constitutive Neuron (Thy1)	[67]	17892
	GCaMP3	500/515	ROSA 26 Gt(ROSA)26Sortm1(CAG-FP)	Conditional (STOPfl/fl)/ whole body (CAG)	[68, 69]	28764 29043 14538
			Omp (Null/KO) Omptm1(FP)	Constitutive. Olfactory neurons (Omp)	[70]	29581
			Unknown Tg(Thy1-FP)	Constitutive Neuron (Thy1)	[67]	28277 17893 29860
			Unknown Tg(tetO/Prnp-FP)	Conditional. DA neuron (TetO)/Brain (Prnp)	[71]	27783
			Unknown Tg(Omp-FP)	Constitutive/olfactory neurons (Omp)	[72]	31780
	GCaMP-GR (GCAMP-mCherry)	1: 500/515 2: 587/610	Random (BAC) Acta2BAC-FP	Constitutive, Sm muscle (Acta2)	[73]	25406
	GCaMP5G/td TomatoFRT/FRT	1: 500/515 2: 554/581	Intergene region downstream Polr2a Polr2aTn(pb-CAG-FP1-IRES-FP2)	Conditional (STOPfl/fl)/ whole body (CAG) tdTomato removable (by FLP)	[40]	24477
	GCaMP6s	500/515	Snap25 (KI) Snap25tm3.1	Constitutive, neuron (KI: Snap25-T2A-FP)	[38]	25111
			ROSA 26 Gt(ROSA)26Sortm96(CAG-FP)	Conditional (STOPfl/fl)/ whole body (CAG)	[38]	28866 24106
			Unknown Tg(Thy1-FP)	Constitutive Neuron (Thy1)	[74]	24275 25776 28278
			Chr 13, between 5710045 and 5710046 Tg(tetO-FP)2	Conditional (TetO)	[41]	24742
			IGs7/TIGRE Igs7tm94.1(tetO-FP)	2xConditional (STOPfl/fl, TetO)	[38]	24104
			IGs7/TIGRE Tg(Camk2a-tTA)1, Igs7tm94.1(tetO-FP)	2xConditional (STOPfl/fl, TetOff); Forebrain (tTA under CaMK2a promoter)	UP	24115
	GCaMP6f	500/515	Unknown Tg(Thy1-FP)	Constitutive Neuron (Thy1)	[74]	25393 24276 24339 28279-28281
			ROSA 26 Gt(ROSA)26Sortm95.1(CAG-FP	Conditional (STOPfl/fl)/ whole body (CAG)	[38]	28865 24105
			IGs7/TIGRE Igs7tm93.1(tetO-FP)	2xConditional (STOPfl/fl, TetO)	[38]	24103 24108
			IGs7/TIGRE Igs7tm148.1(tetO-FP,CAG-tTA2)	1xConditional (STOPfl/fl), TetOff (TetO/tTA2)	[44]	30328
			IGs7/TIGRE, ROSA 26 Igs7tm93.1(tetO-FP), Gt(ROSA)26Sortm5(ACTB-tTA)	1xConditional (STOPfl/fl TetO for FP, STOPfl/fl ACTB for tTA)	[38]	24107
	Salsa6f (GCAMP6ftdTomato)	1:500/515 2:554/581	ROSA 26 Gt(ROSA)26Sortm1.1(CAG-tdTomato-GCaMP6f)	Conditional (STOPfl/fl)/ whole body (CAG)	[75]	31968
	GCaMP8	500/515	Random (BAC) Tg(PromBAC*-FP) Prom=promoter	Constitutive. Various organs/cell types. Prom: Hcn4, Cdh5, Myh6, Sftpc	UP	33341 33342 28344 32885
	GCaMP8.1	500/515	Random (BAC) Tg(Foxj1BAC-FP)	Constitutive, ciliated epithelial cells in lung airway, trachea, testis and oviduct (FoxJ1)	UP	32888
	GCaMP8.1mVermilion	1: 500/515 2: 587/610	Random (BAC) Tg(Acta2BAC-FP-FP)	Constitutive, smooth muscle cells (Acta2 prom)	UP	32887
	RCaMP1.07	575/602	IGs7/TIGRE Igs7tm143.1(tetO-FP)	2xConditional (STOPfl/fl, TetO)	[76]	30217
			Random (BAC) Tg(Acta2BAC-FP)	Constitutive, smooth muscle (Acta2)	UP	28345
	jRGECO1a	Ex ratio: - : 450/595 Ca: 562/588	Unknown Tg(Thy1-FP)	Constitutive, neurons (Thy1)	[77]	30525-30528 32010

The use of protein reporters for spying in the cell may require the cooperation and/or co-expression of multiple transgene products. For example, simultaneous spying on Ca²⁺ in the nuclear (quasi cytoplasmic) and mitochondrial compartment, Ca²⁺ and ROS in the same compartment, or the use of drug-inducible bipartite linker-based interorganelle contact sensors via Ca²⁺/ROS indicator proteins would require coding for two separate protein constructs. Limiting the expression to a tissue/cell type of interest at a certain time (e.g. to avoid adverse long-term Ca²⁺ chelation effects) is also desirable. A useful strategy for such a scenario is to encode the two reporter proteins in tandem in a transgene downstream of a LoxP-flanked stop codon. Inserted between the tandem reporter protein-coding sequences is either a ribosomal skipping-inducing peptide (_2A) coding region or an internal ribosome entry site (IRES), allowing for separation of the translated proteins. The latter strategy was used for transgenic expression of GCaMP5G and tdTomato in the cytoplasm of Cre-expressing cells with a goal to spy on neuronal activity in a quasi ratiometric manner [40]. The Calcium DoubleSpy mice (see later) have been created using the ribosomal skipping-inducing peptide (_2A) strategy. For rapid expression regulation, rendering the expression tetracycline switchable may also be desirable. This requires the tetracycline-responsive promoter (Tet operator, TetO repeats, TRE, upstream of a minimal promoter) and its transcriptional activator, which either associates with (rtTA, Tet-On) or dissociates from (tTA, Tet-Off) the promoter in a tetracycline dependent manner [43]. An additional feature of the tetracycline-controlled transactivator (tTA, rtTA) is that it contains the activating domain of a strong viral transcriptional (trans)activator protein, VP16, which results in an enhanced/amplified gene expression under the TRE/tTA promoter system [43]. For example, the TIGRE locus has been exploited for creating reporter mouse lines with high levels of reporter gene expression in a Cre (LoxP-flanked STOP codon) and tetracycline-dependent manner. However, these mice required either cross-breeding with or viral delivery of both, Cre and tTA [38]. A second generation of these mouse lines (TIGRE2.0) has been simplified so that tTA is already integrated into the transgenic reporter construct, rendering the TRE/tTA-amplified gene expression simply Cre-dependent [44].

Calcium reporter mouse models

Table 1 documents a collection of reporter mice for [Ca²⁺] in the cytoplasm (Table 1A) and various subcellular compartments (1B). The mouse models are grouped by the subcellular localization of the probe, then by the Ca²⁺ sensor protein, the genomic integration site as well as by the mode and tissue distribution of expression along with the corresponding promoter. Notably, the corresponding publications go back no longer than 15 years, underscoring the novelty and perhaps the increasing accessibility of transgenic spy mouse generation. The tissue/cell type specific targeting (and even in the case of conditional reporters, the spectrum of intended tissue/cell targets) is strongly biased towards cell types that are best studied in their tissue context such as neurons, since they rapidly change their phenotype when kept in primary culture (e.g. cardiac and smooth muscle cells). The subcellular targeting is predominantly cytoplasmic with only a few examples of specific membrane targeting: plasma membrane [45, 46], primary cilia [42] and very recently mitochondrial matrix [47].

Calcium DoubleSpy Mice

To delineate the Ca²⁺ communication among different cellular compartments, simultaneous measurements of [Ca²⁺] in each is needed. Using the Ingenious Targeting Laboratory gene editing services https://www.genetargeting.com/, we have recently established a new dual-spy mouse line that we dubbed as “Calcium DoubleSpy”. The spying targets in this mouse are the two essential [Ca²⁺] dynamics readouts for mitochondrial calcium signaling: [Ca²⁺]_c and [Ca²⁺]_m. For the mitochondrial matrix, where motion artifacts become easily significant at high-resolution imaging, we chose the ratiometric GEM-GECO1 with an N-terminal matrix-targeting peptide mt-GEM-GECO1 [48]; and for the cytosol, we chose the spectrally well separable red fluorescent RCaMP1h [49] (Fig1A). Like the GCaMP5G/tdTomato dual transgenic mice [40], to avoid potential adverse effects of constitutive expression, the DoubleSpy construct was made Cre-inducible via the insertion of a LoxP-flanked ‘neostop’ cassette [50] between the strong synthetic CAG2.0 promoter and the reporter-coding regions (Fig1A). The same transgene carries both the mt-GEM-GECO1 and RCaMP1h coding sequences, with a P2A site in between. The P2A peptide-induced ribosomal skipping results in separated reporter proteins. The vector context is a Rosa26 backbone (iTL pCAG2.0-Rosa26) in order to integrate the transgene into the Rosa26 locus via knock-in (Fig 1B, Table 1). The vector was injected to C57BL/6NJ blastocytes and, after standard selection steps, F1 mice were created and back-crossed to wild type C57BL/6NJ mice and maintained as heterozygotes.

Figure 1. Construction and hepatocyte-specific application of the Calcium DoubleSpy mouse.

A. Scheme showing the intracellular targeting of RCaMP and GEM-GECO conditionally expressed in the cells of the Calcium DoubleSpy mice. B. Genomic integration strategy. C. Genetic strategy and fluorescence recording parameters for RCaMP and GEM-GECO. D. Two-photon in vivo imaging in hepatocytes using the recording parameters listed in C. GEM-GECO is only shown at the longer emission wavelength. Imaging was performed 3 weeks after tail vein injection of AAV8-TGB-Cre that effectively and specifically induces expression of the floxed genes in hepatocytes. The bright spots appearing in both green and red channels represent extracellular autofluorescence that showed nonspecific excitation and emission in the entire range of recording. E. Confocal imaging of RCaMP (left) and mtGEM-GECO (middle) in isolated hepatocytes. Excitation and emission wavelengths were GEM-GECO:Ex:405nm, Em:450–550nm & RCaMP Ex: 561nm EM: 575–750nm. F-G. [Ca²⁺]_c (fura2) and [Ca²⁺]_m (mtRCaMP) were simultaneously monitored in isolated hepatocytes by epifluorescence imaging using the recording conditions summarized in C. F. Cells were treated sequentially with a submaximal dose of vasopressin (LVP,1.5nM), and a maximal dose (HVP, 100nM). G. Cells were incubated in a Ca²⁺-free extracellular medium and were sequentially exposed to thapsigargin (TG, 2µM) to discharge the endoplasmic reticulum Ca²⁺ store. Subsequently, CaCl₂ (Ca²⁺, 2mM) was added back to the extracellular medium to initiate store-operated Ca²⁺ entry. FCCP (5µM) and oligomycin (oligo, 2.5µg/ml) were added to uncouple mitochondria. Measurements were done as described previously [62].

To test the Calcium DoubleSpy mice we focused on the liver. The mice were injected intravenously with hepatocyte-directed adeno-associated virus 8 (AAV8)-TBG-Cre ~3 weeks before the imaging experiments [51]. Upon delivery of Cre to the hepatocytes both of the 2 FPs were expressed in the liver as revealed by in vivo 2P liver imaging (Fig1CD). The subcellular targeting of each FP was validated in isolated hepatocytes by confocal microscopy (Fig1E). For initial testing of the Ca²⁺ monitoring we decided to focus on IP₃-mobilizing agonist-induced [Ca²⁺]_c and [Ca²⁺]_m signaling in primary single hepatocytes. Upon sequential stimulation with suboptimal (1.5nM) and saturating (100nM) vasopressin (VP), RCaMP reported baseline-oscillatory transients followed by a large sustained elevation (black trace). The corresponding mt-GEM-GECO1 ratio showed stepwise incremental responses, and likely reached saturation after maximum VP (Fig 1F). The close temporal coupling between each [Ca²⁺]_c spike and the ensuing [Ca²⁺]_m rise is likely because mitochondrial Ca²⁺ uptake was driven by local high-[Ca²⁺] nanodomains at ER-mitochondria contacts and not by the global [Ca²⁺]_c fluctuations [52]. When only global [Ca²⁺]_c rise was evoked via thapsigargin (TG, SERCA inhibitor)-induced ER Ca²⁺ discharge in the absence of extracellular Ca²⁺, it was only followed by a delayed increase in [Ca²⁺]_m (Fig 1G). The robust store-operated Ca²⁺ entry (SOCE) upon restitution of extracellular [Ca²⁺] was followed by a brisk [Ca²⁺]_m rise (Fig 1G). In turn, addition of a mitochondrial uncoupler (protonophore FCCP with F1F0-ATPase inhibitor oligomycin) to dissipate the driving force for mitochondrial Ca²⁺ uptake, the inner membrane potential (ΔΨm), evoked a decrease in the mt-GEM-GECO1 ratio. Interestingly, the corresponding RCaMP fluorescence also decreased slightly, possibly because SOCE decreased [53]. Collectively, these data demonstrate that the new mouse model might serve as an effective “Double-Spy” for dual cytoplasmic and mitochondrial Ca²⁺ monitoring in the same cell, in vivo and in vitro.

Redox and ROS reporter mice

The generation of mouse models expressing GERIs did not begin until the available sensors were somewhat characterized in cultured & ex-vivo cell models. The first mouse model featured roGFP2 introduced via nuclear injection under the control of the tyrosine-hydroxylase (Thy1) promoter to restrict expression to monoaminergic neurons, with targeting to the mitochondrial matrix in a DJ-1 KO Parkinson’s disease model with established redox perturbations [54]. The model revealed perturbations in single mitochondria specific to dopaminergic neurons. By 2016, two mouse models expressing roGFP1, which has a less negative redox midpoint and enhanced pH stability, were developed; both were under Thy1 control, and targeting was to cytosolic or mitochondrial matrix compartments,. The two models revealed compartment specific and neuron subtype-specific redox differences when mapped.

Beyond neuroscience, four mouse models were developed in other redox-labile tissues. In 2011, Xu et al. used a beta-globulin promoter to target roGFP2 to the cytosol of erythrocytes, perhaps the most redox-specialized cell type, revealing an oxidative shift in erythrocyte ageing [55]. Moreover, two mouse models using roGFP1 were expressed in the cytosol or mitochondrial matrix under the control of the elongation factor 1α promoter, which is expressed in tissues including brain, placenta, lung, liver, kidney, and pancreas & skin [56]. The models revealed mitochondrial, but not cytosolic oxidation in a UV model of skin damage. Generation of a kidney-specific mitochondrial roGFP2 mouse by Galvan et al. reported glucose-induced oxidation of the mitochondrial matrix in a diabetic nephropathy model [57].

By fusing roGFP1/2 with human glutaredoxin (Grx1/Grx2), the response of the probe does not rely on the presence/absence of endogenous enzymes that may vary with disease states, compartment, or cell type. In 2014, Breckwold et al, developed a mouse model with Thy1-driven expression of Grx1roGFP2 in the mitochondrial matrix of monoaminergic neurons, which allowed reporting of rapid physiological single-mitochondrial oxidation events and broader oxidation in neuronal injury models [58]. Subsequently, a pair of mouse models expressing Grx1roGFP2 under the control of the α-myosin heavy chain promoter in the cytosol or matrix of cardiomyocytes were created by Swain and colleagues. These models were used to reveal differential, compartment-specific redox changes to stimulation in whole-heart imaging in a Langendorff perfused model [59]. In all cases where roGFP +/−Grx have been used, the probes have been well-tolerated, suitable for conventional/multiphoton imaging and amenable to in-situ calibration, highlighting the suitability of the GSH:GSSG environment to fluorescent sensing. Conversely, probes adapted to sense specific ROS, such as the roGFP2-Orp1 [60], have only been used in fixed preparations to map oxidant distribution in tissues. Similarly, H₂O₂ sensors based on HyPer have not been used to create a mouse model to date. Currently, roGFP probes equipped with Grx demonstrate ideal properties to monitor spatial and temporal shifts in the intracellular redox environment.

Transgenic mice versus alternative GECI/GERI delivery approaches

In vivo transfection with plasmid DNA is applicable in some rare situations such as for delivery of reporters to striated muscles of the footpad. Viral delivery of GECIs or GERIs is effective in several organs. AAV8 is commonly used for hepatocytes of the liver, whereas AAV9 is employed to infect cardiomyocytes in the heart, myotubes in skeletal muscle or neurons of the brain cortex. In the liver, >90% infection rate of the hepatocytes is achievable. However, this strategy is not applicable for expression of FP reporters specifically in a particular cell type e.g. a subset of neurons. Thus, in these cases, inserting the reporter transgene under the control of the cell type specific driver is the only current solution.

A further consideration is the effect of the different delivery approaches on the health of the target cells and surrounding tissues. For example, in vivo electroporation commonly causes tissue reaction and pain. For viral delivery, adenoviruses represented an early opportunity but this option became controversial because of considerable cellular damage was noticed. AAV are better tolerated than adenovirus but some cytopathic effects are unavoidable.

In those tissues of conditional transgenic mice, which can be effectively infected by AAV, expression of the GECI/GERI can be induced either by viral delivery of the Cre or by crossing the reporter mouse with a mice that has a cell type specific Cre transgene. A potential advantage of the AAV-Cre induction is that it does not require lifelong expression of the GECI/GERI, it can produce the FP reporter in a couple of weeks at various points of life.

Limitations

There are limitations specific to the GECI/GERI transgenic mice, whereas other limitations broadly relevant for all FP reporter technologies. As to the first group, constitutively expressed reporters will produce more fluorescence on homozygous background. However, for conditional expression achieved by cross-breeding with Cre or tetracycline-controlled transactivator protein mice, the heterozygous reporter allele is the practical option because it can be achieved in a single breeding cycle. Another potential problem is that when transgenic mice are produced via microinjection of plasmids into the nuclei, a fraction of the mice are mosaic in the germline. This could create heterogeneity among individual cells in expression of the transgene encoding the reporter. As to the imaging, a main restriction is the lack of deep penetration of visible light in tissues. Multiphoton excitation has increased the depth of recording to a couple 100 µms but imaging deep in tissues from the surface remains difficult.

Introduction of the functional probes may cause perturbations of the endogenous pathways. Furthermore, the size of the reporters should also be considered. The molecular weight of a FP is ~30kDa and the above described GECIs and GERIs include 1 or 2 FPs. Proteins of this size unlikely to cause perturbation as soluble proteins in the cytoplasm or in the lumen of other organelles but GECIs/GERIs are also fused to proteins of specific function to spy the local environment. This arrangement might affect the anchor’s function. In addition, light exposure is always a potential source of cell injury in live fluorescence imaging, and the risk of light-induced tissue damage is even greater when physiological pathways are imaged in vivo in mice.

Future opportunities

The progress in mouse genetic technology has increased the speed and lowered the cost of the generation of new transgenic models. Therefore, more FP based reporters are expected to be tested in transgenic mice in the future. Progress is needed and seems to be achievable with the development of NIR FP reporters [61]. This is important because, long-wavelength light between 650 and 900 nm penetrates the furthest through animal tissue. Also, NIR FP reporters will offer an “additional color” for multiparameter in vivo imaging applications. Multiparameter in vivo imaging will allow simultaneous monitoring of interconnected signaling events e.g. both [Ca²⁺] and redox, or signaling events and their outcome at the level of tissue function, which will greatly facilitate the understanding of their physiological and pathophysiological relationships.

Table 1b.

Reporter mice available for organellar [Ca²⁺]

Spying on [Ca2+]	Reporter	Ex/Em (nm)	Insertion site	Constitutive/Conditional. (cell type)	Source	Deposited Jackson #
PM	mGCaMP3	500/515	ROSA 26 Gt(ROSA)26Sortm2 (CAG-FP)	Conditional (STOPfl/fl)/ whole body (CAG) PM via MARCKS domain.	[45]	30170
PM	Lck-GCaMP6f	500/515	ROSA 26 Gt(ROSA)26Sortm1 (CAG-GCaMP6f)	Conditional (STOPfl/fl)/ whole body (CAG) PM via Lck tag	[46]	29626
Primary cilia	Arl13b-mCherry-GECO1.2	1:500/515 2:587/610	Chr 1, 174611546 Tg(CAG-ARL13B/FP/FP)	Constitutive, primary cilia of inner ear (Arl13b prom and gene)	[42]	30613
Mito matrix	Mt-Cam (4mtD3cpv	Ex ratio: - :420/475 Ca:420/535	ROSA 26 Gt(ROSA)26Sortm1 (CAG-FP)	Conditional (STOPfl/fl)/ whole body (CAG) Mt (COX VIII 4xMTS)	[47]
Cyto & Mito matrix	mt-GEM-GECO1/RCaMP1h	1 Em ratio: - : 395/511 Ca:395/455 2:575/602	ROSA 26 Gt(ROSA)26Sortm1(CAG-FP1-P2A-FP2)	Conditional (STOPfl/fl)/ whole body (CAG) Mt (COX VIII 2xMTS)	This paper

Table 2.

Reporter mice available redox and ROS

Spying on	Location	Reporter	Ex/Em	Insertion site	Constitutive/Conditional (cell type)	Source (PMID)	Deposited Jackson #
Redox	Cyto&Mt	roGFP1	Ex:375 Em:475	Non-specific	Constitutive, ubiquitous (EEF1A1) Crossed with hairless model for skin research	[56]	N/A
Redox	Cyto&Mt	roGFP1	Ex:375 Em:475	Non-Specific	Constitutive, Neuronal (Thy1.2)	[78]	N/A
Redox	Mt	roGFP2	Ex:390 Em:485	Non-Specific	Constitutive, erythrocytes (B-globulin)	[55]	N/A
Redox	Mt	roGFP2	Ex:390 Em:485	Non-Specific	Constitutive (Dopaminergic Neurons) TH promoter	[54]	N/A
GSSG:GSH	Mt	Grx1roGFP2	Ex:390 Em:485	Non-Specific	Constitutive Neuron (Thy1)	[58]	N/A
GSSG:GSH	Cyto&Mt	Grx1roGFP2	Ex:390 Em:485	Non-Specific	Constitutive, Heart (a-myosin heavy chain)	[59]	N/A
H2O2	Cyto	RoGFP2-Orp1	Ex:390 Em:485	Rosa26	Constitutive, Neuronal & Global (Thy1/CMV)	[55]	N/A

Acta2,α2-actin (promoter); Actb, β-actin (promoter); Arl13b, ADP-ribosylation factor-like protein 13b (promoter); BAC, bacterial artificial chromosome; CaMK2a, Ca²⁺-calmodulin-dependent kinase 2a (promoter); Cdh5, cadherin 5 (promoter); COX VIII, cytochrome oxidase subunit VIII; Cx40, connexin 40 (promoter); DA, dopaminergic; Em, λ emission; Ex, λ excitation; FoxJ1, forkhead box J1 (promoter); FP, refers to the respective reporter FP; G, green; Gt, gene trap; Hcn4, hyperpolarization-activated cyclic nucleotide-gated K+ channel 4 (promoter); IG, inter gene (region); KI, knock-in; MTS, mitochondrial (matrix) targeting sequence; Myh6, myosin heavy chain 6 (promoter); Omp, olfactory marker protein (gene/promoter); PC, photoconversion; PM, plasma membrane; Prnp, prion protein (promoter); R, red; tm, targeted mutation; Sftpc, surfactant protein-C (promoter); tetO, tet operator; UP, unpublished; Tg, transgene; Thy1, thymus cell antigen 1 (promoter)

Abbreviations

AAV

Adeno Associated Virus

Acta2

α2-actin (promoter)

Actb

β-actin (promoter)

Arl13b

ADP-ribosylation factor-like protein 13b (promoter)

BAC

bacterial artificial chromosome

[Ca²⁺]_c

cytoplasmic [Ca²⁺]

[Ca²⁺]_m

mitochondrial matrix [Ca²⁺]

CaMK2a

Ca²⁺-calmodulin-dependent kinase 2a (promoter)

Cdh5

cadherin 5 (promoter)

Cyto

Cytoplasm

COX VIII

cytochrome oxidase subunit VIII

Cx40

connexin 40 (promoter)

Ex

λ Excitation

Em

λ Emission

DA

dopaminergic

DHR

Dihydrorhodamine

FoxJ1

forkhead box J1 (promoter)

GSH

Glutathione tripeptide

Gt

gene trap

Hcn4

hyperpolarization-activated cyclic nucleotide-gated K+ channel 4 (promoter)

HOCl

Hypochlorous acid

IMM

inner mitochondrial membrane

IMS

intermembrane space

IG

inter gene (region)

KI

knock-in

KO

knock-out

mt

mitochondrial matrix

mPTP

mitochondrial permeability transition pore

MTS

mitochondrial (matrix) targeting sequence

mtCU

Ca²⁺ uniporter

Myh6

myosin heavy chain 6 (promoter)

NIR

Near Infra Red

O₂^•−

superoxide

OMM

outer mitochondrial membrane

Omp

olfactory marker protein (gene/promoter)

PC

photoconversion

PM

plasma membrane

Prnp

prion protein (promoter)

Tm

targeted mutation

ONOO^•−

Peroxinitrite

ROS

Reactive Oxygen Species

Sftpc

surfactant protein C (promoter)

tetO

tet operator

UP

unpublished

Tg

transgene

Thy1

thymus cell antigen 1 (promoter)

VDAC

Voltage Dependent Anion-selective Channel

WT

wild type