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Cited by 1,114Advanced Fluorescence Microscopy TechniquesNeuroscience and Neuropharmacology ResearchPhotoreceptor and optogenetics research

Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance

Open access
Madisen, Linda, Garner, Aleena R., Shimaoka, Daisuke, Chuong, Amy S., Klapoetke, Nathan C., Li, Lu, van der Bourg, Alexander, Niino, Yusuke +18 more·2015·Neuron
DOI 10.1016/j.neuron.2015.02.022PMC PMC4365051
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Abstract

Summary An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems, and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.

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Written from the paper’s full text.

TL;DR

The paper develops intersectional transgenic mouse tools for targeting neural sensors and effectors with improved specificity and expression, centered on a new retargetable TIGRE locus and Cre/tTA-dependent transcriptional amplification. It validates these lines functionally for voltage, calcium, glutamate, and optogenetic tools, with TIGRE reporters generally outperforming Rosa-CAG reporters.

Key findings

  • The authors generated 17 reporter lines, 4 driver lines, and 10 AAVs, including Cre/Flp and Cre/Dre double-recombinase systems and a new TIGRE docking locus flanked by HS4 insulators. TIGRE-based Ai62 tdTomato fluorescence per cell was 2–4 fold higher than the Rosa-CAG Ai14 reporter in FACS analysis of cortical neurons.
  • Intersectional Cre/Flp and Cre/Dre targeting increased specificity: Slc32a1-IRES-Cre;Pvalb-2A-Flpo;Ai65 mice showed tdTomato only in Slc32a1+/Pvalb+ cells, with no reporter expression without both drivers, whereas Pvalb-2A-Cre;Ai14 mice also labeled cortical layer 5 pyramidal neurons and other non-interneuron cells. Cre showed low-frequency promiscuous recombination at Rox sites, producing occasional tdTomato cells in Cre+/Dre−/Ai66+ controls.
  • Functional validation showed strong performance: Ai78 VSFP-Butterfly 1.2 gave visual cortex S/N ratios of 11.8 in an example mouse and 12.1 ± 2.7 SEM in five other mice, not statistically different from in utero electroporation values (S/N = 5.54 ± 0.44, n = 3 mice; p = 0.05, Mann-Whitney test). Ai93 GCaMP6f visual responses reached ΔF/F of 500% or higher, and Ai93 detected more active neurons than Ai95 at 120 μm (102 ± 18 vs 35 ± 5) and 300 μm (129 ± 11 vs 18 ± 5; n = 3 mice each, p = 4.6 × 10−5 between genotypes).
  • Ai79 TIGRE-Jaws produced strong red-light-induced suppression of spontaneous firing in vivo (n = 23 units from 2 mice; 3 units showed no change), comparable to virally expressed Jaws, while Ai57 Rosa-CAG-Jaws showed smaller photocurrents in slices.

Methods

  • Transgenic mice were generated by homologous recombination into endogenous loci, including Rosa26, TIGRE, and cell-type-specific loci, often followed by Flp-mediated RMCE. The study used Cre/Flp, Cre/Dre, and Cre/tTA intersectional strategies, plus recombinant AAVs expressing recombinases and doubly regulated reporters.
  • Reporter expression was assessed by native fluorescence epifluorescence or confocal imaging, FACS of single-cell suspensions, and in situ hybridization. Functional assays included hippocampal slice voltage imaging, in vivo wide-field voltage imaging through thinned skull, in vivo two-photon calcium imaging in visual and somatosensory cortex, and slice/in vivo electrophysiology for optogenetic silencing.
  • Sample sizes for key functional comparisons included n = 3 mice each for Ai95 vs Ai93 GCaMP6f imaging, n = 13 cells in Ai57, n = 17 in Ai79, and n = 12 in Ai35 for Jaws slice electrophysiology, and n = 23 units from 2 mice for in vivo Jaws recordings. Experiments followed NIH, UK, Swiss, and New Zealand animal welfare guidelines, with local IACUC or equivalent approvals.

Limitations

  • The paper states that expression varied significantly among sensors and effectors even with the same TIGRE or Rosa26 configuration, likely due to intrinsic protein properties, so stable transgenic expression remains unpredictable and must be examined experimentally in vivo.
  • The authors report that several knock-in tTA driver lines showed no or low tTA-induced reporter expression, possibly due to epigenetic silencing of the Tet system, limiting the available tTA driver repertoire; only Camk2a-tTA and ROSA26-ZtTA were validated as effective TIGRE drivers.
  • The paper notes that intersectional transgenic strategies require laborious breeding to generate triple transgenic mice, and that Cre can promiscuously recombine Rox sites at low frequency. It also cautions that animals expressing engineered proteins should be monitored for unexpected adverse effects, and that reporter expression patterns can vary depending on the tTA line used, with ROSA26-ZtTA driving fewer cells in some subcortical regions.

Model-written summary. Check it against the paper before relying on it.

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Registry

Crossref · 10.1016/j.neuron.2015.02.022

Cites

70 of 70 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 62 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws.

    “High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws.”
    Quote in the paperSubstantial claim
  • Findingown data

    Cre and Flp efficiently restricted transgene expression to cell populations with overlapping patterns of driver expression.

    “Cre and Flp (both Pvalb-2A-Flpe and Pvalb-2A-Flpo) efficiently restricted transgene expression to cell populations with overlapping patterns of driver expression.”
    Quote in the paperSubstantial claim
  • Findingown data

    The authors established a new retargetable genomic locus, TIGRE, which allowed generation of a large set of Cre/tTA dependent reporter lines expressing fluorescent proteins, calcium, voltage, or glutamate indicators, and optogenetic effectors.

    “established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors”
    Quote in the paperSubstantial claim
  • Findingown data

    The authors generated multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity using intersectional strategies.

    “Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity.”
    Quote in the paperSubstantial claim
  • Findingreasoning

    The authors created a series of TIGRE reporter lines that show high-level expression of novel calcium, voltage and glutamate sensors and optogenetic effectors.

    “Since the third strategy resulted in the most strongly enhanced transgene expression, we created a series of TIGRE reporter lines that show high-level expression of novel calcium, voltage and glutamate sensors and optogenetic effectors.”
    Quote in the paperSubstantial claim
  • May combine two claims. 44
  • Classified as probably not a substantial claim. 11

  • May combine two claims.

    Findingown data

    The paper introduces 17 reporter lines, 4 driver lines, and 10 AAVs.

    “The complete list of new intersectional transgenic mouse lines and AAVs introduced in this paper (17 reporter lines, 4 driver lines and 10 AAVs) is shown in Table 1.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    AAV-Flpo and AAV-Dre induced recombination comparable to AAV-Cre, whereas the recombination efficiency of AAV-Flpe was significantly lower.

    “Consistent with previous reports (Fenno et al., 2014; Kranz et al., 2010), AAV-Flpo and AAV-Dre induced recombination comparable to AAV-Cre, whereas the recombination efficiency of AAV-Flpe was significantly lower.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Fenno et al., 2014 source

      Not checked in the free run.

    • cites Kranz et al., 2010 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Flp and Dre viruses exhibited high specificity for FRT and Rox sites, respectively, whereas AAV-Cre recombined Rox sites at low frequency, with recombination most prevalent at the injection site, indicating increased promiscuity with high levels of Cre.

    “Flp and Dre viruses exhibited high specificity for FRT and Rox sites, respectively, whereas AAV-Cre recombined Rox sites at low frequency, with recombination most prevalent at the injection site, indicating increased promiscuity with high levels of Cre.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Trib2-2A-CreERT2;Ai14 mice, enriched expression was seen in layer 5a cortical neurons, but also strong and widespread fluorescence in vasculature.

    “Likewise, in Trib2-2A-CreERT2;Ai14 (Trib2 is tribbles homolog 2) we saw enriched expression in layer 5a cortical neurons, but also strong and widespread fluorescence in vasculature.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All reporters demonstrated the expected specificity of expression when paired with double or triple drivers, expanding the repertoire of viral tools for highly specific and versatile genetic control.

    “All reporters demonstrated the expected specificity of expression when paired with double or triple drivers, thereby further expanding the repertoire of viral tools that can be used for highly specific and versatile genetic control (Fig. S4E-G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GCaMP6s expression in the TIGRE allele Ai94 was stronger than the Rosa-CAG allele Ai96 and also stronger than the Snap25-2A-GCaMP6s line, which like Ai96 showed no basal level fluorescence.

    “In addition, GCaMP6s expression in the TIGRE allele Ai94 was not only stronger than the Rosa-CAG allele Ai96 (Fig. 3B), but also stronger than the Snap25-2A-GCaMP6s line which, like Ai96, showed no basal level fluorescence (data not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Nxph4-2A-CreERT2;Ai14 mice, sparse cortical layer 6b expression was observed, but also significant expression in small, possibly glial cells.

    “In Nxph4-2A-CreERT2;Ai14 (Nxph4 is neurexophilin 4), we observed sparse cortical layer 6b expression, but also significant expression in small, possibly glial cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Pvalb-2A-Cre;Ai14 mice, but not in the triple Tg mice, expression was found in cortical layer 5 pyramidal neurons and other non-interneuron cells, presumably arising from low-level or transient expression of the Pvalb gene.

    “In those mice, but not in the triple Tg mice, we found expression in cortical layer 5 pyramidal neurons and other non-interneuron cells, presumably arising from low-level or transient expression of the Pvalb gene (Fig. S2B, C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The authors generated a Snap25-2A-GCaMP6s knock-in line that expresses GCaMP6s pan-neuronally and independently of any driver line.

    “To this end, we generated a Snap25-2A-GCaMP6s knock-in line, which expresses a calcium indicator GCaMP6s (Chen et al., 2013) pan-neuronally and independently of any driver line (Table 1).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Chen et al., 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    Snap25-LSL-2A-GFP showed stronger GFP fluorescence than the Rosa-CAG based Ai3-EYFP reporter.

    “Snap25-LSL-2A-GFP also showed stronger GFP fluorescence than the Rosa-CAG based Ai3-EYFP reporter (Madisen et al., 2010) (data not shown).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Madisen et al., 2010 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Control mice lacking Cre showed no reporter expression, but occasional tdTomato positive cells were observed in Cre+/Dre-/Ai66+ mice.

    “Control mice lacking Cre showed no reporter expression, but occasional tdTomato positive cells in Cre+/Dre-/Ai66+ mice were observed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was no significant difference in the numbers of expressing cells either between different lines or across ages.

    “There was no significant difference in the numbers of expressing cells either between different lines or across ages.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    When regulated by some more specific Cre drivers, although general expression patterns are similar between Rosa26 and TIGRE reporters, variations can occur depending on which tTA line is used for activation, with ROSA26-ZtTA tending to drive expression in fewer cells compared to Ai14, especially in subcortical regions.

    “When regulated by some more specific Cre drivers, however, we found that although general expression patterns are similar between Rosa26 and TIGRE reporters, variations can occur depending on which tTA line is used for activation, with ROSA26-ZtTA tending to drive expression in fewer cells compared to Ai14, especially in subcortical regions (Fig. S7B-D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Strong expression and proper localization to the plasma membrane were seen in lines expressing membrane proteins VSFPB (Ai78) and Jaws (Ai79).

    “We saw strong expression and proper localization to the plasma membrane in lines expressing membrane proteins VSFPB (Ai78, Fig. 4B) and Jaws (Ai79, Fig. 4F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The Nr4a2-SA-IRES-Dre;Emx1-IRES-Cre;Ai66 triple Tg mice showed specific, dense expression in claustrum and endopiriform nucleus, consistent with adult brain areas known to express both Emx1 and Nr4a2.

    “The resulting triple Tg mice showed specific, dense expression in claustrum and endopiriform nucleus (Fig. 1C), consistent with adult brain areas known to express both Emx1 and Nr4a2.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Emx1 and Nr4a2 are expressed in claustrum and endopiriform nucleus.

  • May combine two claims.

    Findingown data

    Again, no reporter expression was observed in the absence of Cre, but sparse tdTomato positive cells were detected in Cre+/Dre-/Ai66+ mice.

    “Again, we observed no reporter expression in the absence of Cre but did detect sparse tdTomato positive cells in Cre+/Dre-/Ai66+ mice (Fig. S3B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Scnn1a-Tg3-Cre;Camk2a-tTA;Ai93 triple Tg mice, GCaMP6f-labeled layer 4 neurons exhibited low baseline fluorescence in vivo, while visual stimuli evoked responses with ΔF/F reaching 500% or higher.

    “In Scnn1a-Tg3-Cre;Camk2a-tTA;Ai93(TITL-GCaMP6f) triple Tg mice (Fig. 6A-C), GCaMP6f-labeled layer 4 neurons exhibited low baseline fluorescence in vivo (Fig. 6A), while visual stimuli evoked responses with ΔF/F reaching 500% or higher (Fig. 6A-C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In response to optimally placed visual stimuli, visual cortex gave S/N ratios of 11.8 in the example mouse, and similar values in five other mice (S/N = 12.1 ± 2.7 SEM).

    “In response to optimally placed visual stimuli, visual cortex gave S/N ratios of 11.8 in the example mouse (Fig. 5J), and similar values in five other mice (S/N = 12.1 ± 2.7 SEM).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Ai79 mice, 632-nm red light induced strong hyperpolarizing currents in Jaws-expressing neurons and effectively blocked depolarization-induced spiking.

    “Red light illumination (632 nm) induced strong hyperpolarizing currents in Jaws-expressing neurons in Ai79 mice and effectively blocked depolarization-induced spiking”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At every irradiance tested, Ai79 photocurrents matched those from virally expressed Jaws, whereas Ai57 cells had significantly smaller photocurrents.

    “Photocurrents were comparable between Ai79 and virally expressed Jaws at all irradiance levels tested, but were significantly smaller in Ai57 cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ai95 mice had significantly fewer detectable active neurons than Ai93 mice, with the difference most pronounced at greater depths.

    “the number of active neurons observable was significantly lower in Ai95 mice compared to Ai93 mice, especially deeper into the tissue”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    These responses could be observed both as increased acceptor signals and as decreased donor signals.

    “These responses could be observed both as increased acceptor signals and as decreased donor signals (Akemann et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Akemann et al., 2012 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Visual cortex also responded to the onset of air puffs, whether delivered towards or away from the whiskers, indicating that these responses were due to the sound of the air puffs, specifically the first puff in the train.

    “Visual cortex, however, also responded to the onset of air puffs, whether they were delivered towards the whiskers (Fig. 5K) or away from them (Fig. 5L), indicating that these responses were due to the sound of the air puffs, and specifically by the first puff in the train.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the two-way ANOVA of active neuron counts, genotype differed significantly, depth did not, and there was no interaction.

    “Two-way ANOVA revealed a significant difference between genotypes, but not by depths and with no interaction”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The increase was sub-linear, consistent with temporal summation and frequency dependent depression of synapses from medial perforant path to dentate granule cells.

    “The increase was sub-linear, consistent with the temporal summation and well-known frequency dependent depression of the synapses from medial perforant path to dentate granule cells (Petersen et al., 2013).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Petersen et al., 2013 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Auditory cortex responded approximately equally to air puffs, whether delivered towards or away from the whiskers, and did not respond to visual stimuli.

    “Finally, auditory cortex responded approximately equally to air puffs, whether they were delivered towards or away from the whiskers (Fig. 5Q, R), and did not respond to visual stimuli (Fig. 5P).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In contrast, somatosensory cortex responded only to air puffs delivered to the whiskers, and not to visual stimuli or to the sounds of the air puffs.

    “In contrast, somatosensory cortex responded only to the air puffs delivered to the whiskers (Fig. 5N), and not to visual stimuli or to the sounds of the air puffs (Fig. 5M, O).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    VSFP transients were readily resolved above baseline noise in single sweeps, with amplitude approximately 3-4 times baseline noise.

    “We readily resolved VSFP transients above baseline noise in single sweeps (amplitude ∼3-4× baseline noise, not shown).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The authors conducted in vivo two-photon imaging through a cranial window over visual cortex in head-fixed, running GCaMP6 reporter mice, and were able to simultaneously image hundreds of neurons relatively deep in the cortex (~300 μm below the pia surface, corresponding to visual cortical layer 4).

    “We conducted in vivo two-photon imaging (Andermann et al., 2011; Bonin et al., 2011) through a cranial window over visual cortex in head-fixed, running GCaMP6 reporter mice, and were able to simultaneously image hundreds of neurons relatively deep in the cortex (∼300 μm below the pia surface, corresponding to visual cortical layer 4).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Andermann et al., 2011 source

      Not checked in the free run.

    • cites Bonin et al., 2011 source

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  • May combine two claims.

    Findingown data

    Both Rosa26 and TIGRE homozygous transgenic mice are fertile.

    “We found that both Rosa26 and TIGRE homozygous Tg mice are fertile”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The light-induced inhibition of neuronal firing in Ai79 mice was intensity-dependent and comparable to virally expressed Jaws.

    “Light delivery through the fiber tip inhibited neuronal firing in a light intensity-dependent manner in these mice, comparable to the performance of virally-expressed Jaws”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Targeting genetically encoded sensors and effectors to specific cell types is an increasingly powerful approach for studying brain circuits.

    “An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Huang and Zeng, 2013 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Important efforts have been made to develop transgenic intersectional approaches, most successfully with the combination of Cre and Flp site-specific recombinases.

    “Important efforts have been made to develop transgenic intersectional approaches, most successfully with the combination of Cre and Flp site specific recombinases (SSRs) (Dymecki and Kim, 2007; Dymecki et al., 2010; Kranz et al., 2010; Ray et al., 2011; Robertson et al., 2013).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Dymecki and Kim, 2007

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    • cites Dymecki et al., 2010 source

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    • cites Kranz et al., 2010 source

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  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Premisecitation

    The ongoing development of increasingly effective sensors and effectors offers extraordinary opportunities for studies of neuronal interactions and functions.

    “The ongoing development of increasingly effective sensors and effectors offers extraordinary opportunities for studies of neuronal interactions and functions (Fenno et al., 2011; Huang and Zeng, 2013; Knopfel, 2012).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Fenno et al., 2011 source

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    • cites Huang and Zeng, 2013 source

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    • cites Knopfel, 2012 source

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  • Classified as probably not a substantial claim.

    Premisecitation

    In mice, the Cre/lox recombination system is the most widely used approach to access specific cell types, utilizing gene promoters or loci with specific expression patterns.

    “In mice, the Cre/lox recombination system is the most widely used approach to access specific cell types, utilizing gene promoters or loci with specific expression patterns (Gerfen et al., 2013; Gong et al., 2007; Madisen et al., 2010; Taniguchi et al., 2011).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Gerfen et al., 2013 source

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    • cites Gong et al., 2007 source

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    • cites Madisen et al., 2010 source

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  • May combine two claims.

    Interpretationown data

    This result suggests that while Dre specifically recombines Rox sites over Lox sites, Cre is slightly promiscuous towards Rox sites.

    “This result suggests that while Dre specifically recombines Rox sites over Lox sites, Cre is slightly promiscuous towards Rox sites.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Premiseown data

    Snap25 is pan-neuronal and is among the most highly transcribed genes in the brain based on microarray data analysis.

    “It is pan-neuronal and is among the most highly transcribed genes in the brain based on microarray data analysis (data not shown).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The tetracycline-regulated expression system has been used in mice for inducible expression of genes from the TRE promoter in response to tTA or rtTA activation.

    “The tetracycline (Tet)-regulated expression system has been used in mice for inducible expression of genes from the TRE (tetracycline response element, also called tetO) promoter in response to tTA (Tet-Off) or rtTA (Tet-On) activation (Garner et al., 2012; Gossen and Bujard, 1992; Mayford et al., 1996; Reijmers et al., 2007; Urlinger et al., 2000).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Garner et al., 2012 source

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    • cites Gossen and Bujard, 1992 source

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    • cites Mayford et al., 1996 source

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  • May combine two claims.

    Interpretationown data

    The TIGRE-targeted reporter is advantageous over Rosa26-CAG reporter in more robust transgene expression and the potential for additional specificity mediated through restricted tTA expression.

    “The above comparison indicates that the TIGRE-targeted reporter is advantageous over Rosa26-CAG reporter in more robust transgene expression and the potential for additional specificity mediated through restricted tTA expression.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    VSFP-Butterfly 1.2 yields strong voltage signals in mouse sensory cortex, both under anesthesia and in wakefulness.

    “VSFP-Butterfly 1.2 yields strong voltage signals in mouse sensory cortex, both under anesthesia and in wakefulness (Akemann et al., 2012; Carandini et al., 2015; Scott et al., 2014).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Akemann et al., 2012 source

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    • cites Carandini et al., 2015 source

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    • cites Scott et al., 2014 source

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  • May combine two claims.

    Premisenone given

    The new TIGRE allele differs from the Rosa26 allele in three key aspects: genomic location, use of the TRE promoter instead of the CAG promoter, and the flanking insulators.

    “The new TIGRE allele differs from the Rosa26 allele in three key aspects: genomic location, use of the TRE promoter instead of the CAG promoter, and the flanking insulators.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The interplay between Cre, tTA and reporter alleles among different cells in different mouse lines may not always be predictable, so reporter expression needs to be carefully assessed in new lines to ensure their applicability.

    “These findings indicate that the interplay between Cre, tTA and reporter alleles among different cells in different mouse lines may not always be predictable, thus reporter expression needs to be carefully assessed in new lines to ensure their applicability.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Previous work showed that TRE-driven genes targeted to the mouse TIGRE locus could be expressed in most tissues in the presence of tTA.

    “Previous work showed that TRE-driven genes targeted (via a retrovirus) to the mouse TIGRE locus, situated on Chromosome 9 between the AB124611 (HIDE1) and Carml loci, could be expressed in most tissues in the presence of tTA (Zeng et al., 2008).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Zeng et al., 2008 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GCaMP6 variants constitute the latest generation of genetically encoded calcium indicators with fundamentally improved sensitivity and performance over previous generations, e.g. GCaMP3 and GCaMP5.

    “GCaMP6 variants constitute the latest generation of genetically encoded calcium indicators (GECIs) (Chen et al., 2013) with fundamentally improved sensitivity and performance over previous generations, e.g. GCaMP3 and GCaMP5.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chen et al., 2013 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Jaws is the first red-light-drivable optogenetic inhibitor and was engineered from H. salinarum strain Shark cruxhalorhodopsin.

    “Jaws is the first red light drivable optogenetic inhibitor, engineered from H. salinarum (strain Shark) cruxhalorhodopin (Chuong et al., 2014).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chuong et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Ratiometric imaging with FRET-based calcium indicators such as yellow cameleon has reduced sensitivity to motion artifacts and permits quantification of calcium concentration levels.

    “Ratiometric imaging using FRET-based calcium indicators, such as yellow cameleon (YC) indicators (Horikawa et al., 2010; Nagai et al., 2004; Yamada et al., 2011), is another approach that has the advantage of reduced sensitivity to motion artifacts and essentially permits quantification of calcium concentration levels.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Horikawa et al., 2010 source

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    • cites Nagai et al., 2004 source

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    • cites Yamada et al., 2011 source

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    Premisecitation

    Relative to eNpHR3.0, Arch, and ArchT, Jaws has robust red-light responses, high sensitivity, large photocurrents, and fast kinetics.

    “Compared with other photo-activated inhibitors such as eNpHR3.0, Arch or ArchT, Jaws responds robustly to red light with high sensitivity, large photocurrent and fast kinetics”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Chuong et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Jaws permits deeper tissue penetration and more effective silencing of large volumes.

    “and thus enables better tissue penetrance and more effective optogenetic silencing of large volumes (Chuong et al., 2014).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Chuong et al., 2014 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Breeding with homozygous alleles or double transgenic by double transgenic crosses should improve efficiency.

    “So using homozygous alleles and/or double Tg × double Tg breeding schemes will increase breeding efficiency.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductreasoning

    Mouse experiments were conducted according to NIH, UK, Swiss, and New Zealand guidelines and approved by the relevant institutional committees.

    “All experimental procedures related to the use of mice were conducted according to NIH guidelines, the UK Animals Scientific Procedures Act (1986), the guidelines of the Veterinary Office of Switzerland, and the New Zealand Animal Welfare Act (1999).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The reporter line collection should enable diverse observation and manipulation of cell types, with advantages over viral methods for broad, uniform, and long-lasting monitoring and manipulation of neuronal activity.

    “The collection of reporter lines should facilitate various ways of observing and manipulating cell type functions, having advantage over viral expressions in large-area, uniform, and long-term monitoring and manipulation of neuronal activities.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The observed variation in protein expression is most likely due to intrinsic protein properties because the proteins originate from diverse non-mammalian species.

    “We think this variation is most likely due to intrinsic protein properties, as they originate from diverse non-mammalian species.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Stably integrated transgenes in mice give unpredictable expression and must always be checked experimentally in vivo.

    “Therefore expression in mice as a stably-integrated transgene is unpredictable and always needs to be examined experimentally in vivo.”
    Quote in the paperSubstantial claim
53 of 53

  • The paper relies on this without support.

    Cell types are rarely defined by single genes but by intersectional expression of multiple genes.

    “Fundamentally, cell types are rarely defined by single genes, but rather by intersectional expression of multiple genes.”

  • The paper relies on this without support.

    High-level expression of sensors and effectors is required for their practical utility.

    “One issue with the practical utility of these tools is that they require high-level expression in cell populations of interest.”

  • The paper relies on this without support.

    Viral infection of GCaMP leads to unhealthy cells whose nuclei become filled with fluorescence within weeks.

    “This result contrasts the common observation associated with viral infection of GCaMP where transgene expression continues to rise and leads to unhealthy cells whose nuclei become filled with fluorescence within weeks.”

  • The authors state this as an assumption.

    The ROSA:LNL:tTA and ROSA26-ZtTA lines have Cre-dependent ubiquitous expression.

    “The other two tTA lines are both targeted to the Rosa26 locus and contain floxed-stop cassettes, and are thus presumed to have Cre-dependent ubiquitous expression.”

  • The paper relies on this without support.

    Intersectional approaches have not been widely used in functional studies due to the limited number of validated transgenic tools available.

    “However, thus far, intersectional approaches have not been widely used in functional studies, due to the limited number of validated transgenic tools available.”

  • The paper relies on this without support.

    Nr5a1-Cre drives specific expression in cortical layer 4 neurons.

    “For a common Cre line we used Nr5a1-Cre, which drives specific expression in cortical layer 4 neurons.”

  • The paper relies on this without support.

    Emx1 and Nr4a2 are expressed in claustrum and endopiriform nucleus.

    “consistent with adult brain areas known to express both Emx1 and Nr4a2.”

  • The paper relies on this without support.

    Slc32a1 and Pvalb overlap in certain cells.

    “consistent with Slc32a1+/Pvalb+ expression pattern”

  • The paper relies on this without support.

    Thy1.2 promoter-driven transgene expression is strongly position dependent, necessitating a screen of multiple founder lines.

    “Expression of transgenes driven by the Thy1.2 promoter is strongly position dependent, necessitating a screen of multiple founder lines to find potentially useful ones.”

  • The paper relies on this without support.

    Adding Cre-dependent control to a multi-copy transgene is problematic because Cre could induce recombination within and between copies, reducing copy numbers and causing variability.

    “Furthermore, adding Cre-dependent control, e.g. a floxed-stop cassette, to a multi-copy transgene is problematic because Cre could then induce recombination both within and between the different copies, resulting in reduced transgene copy numbers and variability of transgene expression among different cells.”
10 of 10

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Authors
Madisen, L., Garner, A. R., Shimaoka, D., Chuong, A. S., Klapoetke, N. C., Li, L., van der Bourg, A., Niino, Y., Egolf, L., Monetti, C., Gu, H., Mills, M., Cheng, A., Tasic, B., Nguyen, T. N., Sunkin, S. M., Benucci, A., Nagy, A., Miyawaki, A., Helmchen, F., Empson, R. M., Knöpfel, T., Boyden, E. S., Reid, R. C., Carandini, M., & Zeng, H.
Affiliations
  • Allen Institute for Brain Science (US)
  • University College London (GB)
  • MIT Media Lab (US)
  • RIKEN Center for Brain Science (JP)
  • University of Zurich (CH)
  • Mount Sinai Hospital (CA)
  • University of Otago (NZ)
  • Imperial College London (GB)
Corresponding author
Zeng, Hongkui
ORCID
Venue
Neuron
Publisher
Elsevier BV
Published
2015-03
Volume
85
Issue
5
Pages
942-958
ISSN
0896-6273 (ISSN-L)
DOI
10.1016/j.neuron.2015.02.022
PMC
PMC4365051
OpenAlex
W1975032719
Semantic Scholar
bd6a00f49932432d80cede83a881ad349257d6d5
Cited by
1,114
References
70
Citations by year
2026: 64 · 2025: 90 · 2024: 108 · 2023: 110 · 2022: 118 · 2021: 107 · 2020: 137 · 2019: 116 · 2018: 104 · 2017: 80 · 2016: 56 · 2015: 24
FWCI
53.24
Type
journal article
Language
en
Open access
Licence
Locations
  • Neuron (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (acceptedVersion)
  • Queen Mary Research Online (Queen Mary University of London) (submittedVersion, cc-by-nc-nd)
  • DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
  • UCL Discovery (University College London) (submittedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (submittedVersion)
Funders
  • GlaxoSmithKline
  • Wellcome Trust (095669)
  • Animal Welfare Institute
  • National Institutes of Health
  • National Institute of Mental Health (MH085500, R01MH085500)
  • National Institute on Drug Abuse (DA028298, R01DA028298)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Photoreceptor and optogenetics research
Keywords
transgenic mice, genetically encoded calcium indicators, genetically encoded voltage indicators, neuronal activity monitoring, brain circuits, viral vectors
MeSH terms
Animals, Hippocampus, Mice, Transgenic, Neurons, Organ Culture Techniques, Visual Cortex, Gene Targeting, Integrases, Mice, Optogenetics

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

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