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Optogenetic, transient-sensing and chemogenetic mouse models available from The Jackson Laboratory. Jason Beckwith, Stephen F. Rockwood, Michael Sasner The Jackson Laboratory, Bar Harbor, Maine 04609 USA Program/Poster#: 558.07 / MMM57 ABSTRACT OPTOGENETICS The Jackson Laboratory (JAX) Mouse Repository distributes mouse lines with optogenetic and transient-sensing (calcium-, voltage-, glutamate-) technologies. Opsins are light-activated proteins that alter membrane potential in neurons, so that stimulation with light allows rapid control of neuronal activity. Several mouse lines express improved/optimized opsins fused to fluorescent proteins. These include mice with channelrhodopsin expression directed by specific promoters. Additional control is available in mice with Creor Tet-dependent expression of channelrhodopsin or halorhodopsin variants. Variants of GCaMP fluoresce in response to calcium binding and serve as an indicator of neuronal activation. These include the Thy1-promoter driven GCaMP3, GCaMP6f or GCaMP6s transgenic lines, the Tet-dependent GCaMP6f or GCaMP6s transgenic lines and the Cre-dependent GCaMP3, GCaMP5, GCaMP6f or GCaMP6s mouse lines. Several strains utilize both Cre-lox and Tet-On/-Off functionality. Removal of a floxed-STOP allows Tet-dependent expression of channelrhodopsin (Chronos/EGFP), halorhodopsin (Jaws/EGFP), GCaMP6s, GCaMP6f or FRET chromophores sensitive to calcium (YCX2.60), voltage (VSFPB1.2) or glutamate (iGluSnFR). This set features models created by the Allen Institute for Brain Science, the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project (Janelia/HHMI), Duke/MIT and several others. Transgenic lines from the Cornell Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus) are designed for combinatorial crosses enabling the coexpression of sensors and effectors, or red and green calcium sensors (e.g., RCaMP and GCaMP8). Designer receptors exclusively activated by designer drugs (DREADDs) are mutant G-protein coupled receptors activated by the pharmacologically-inert molecule clozapine-N-oxide. Several chemogenetic strains have Cre-, Tetand/or FLP-inducible expression of M3Dq, M4Di or M3Ds. The JAX Mouse Repository - a centralized facility for the development, rederivation, cryopreservation and distribution of mouse models to the international biomedical research community - adds hundreds of new strains annually. The JAX Mouse Repository is supported by NIH, The Howard Hughes Medical Institute and several private charitable foundations. A. INTRODUCTION OPTOGENETICS: control of cellular functions in genetically modified cells using opsins - transmembrane, retinal-binding proteins that combine a light-sensitive domain with an ion channel or pump. Upon absorption of light, the protein is activated and provides ion transport, membrane potential alteration and sensory functions to bacteria. By exogenously expressing light-activated proteins that alter membrane potential in neurons, addition or removal of specific wavelengths of light can be used for rapid control of neuronal activity. B. OPSINS www.jax.org/donate-a-mouse Donating a Strain to The Jackson Laboratory GCaMP2 α-SMA GCaMP3 / mCherry Hcn4 COMMON NAME Acta2BAC-GCaMP2 smooth muscle (incl. vessels) GCaMP8 BAC Acta2 -GCaMP-GR HCN4-GCaMP8 heart, brain, spinal cord JAX# 025405 028344 Olf. bulb M1 Piriform Amygdala S1 Hippocamp Thalamus Hypothal V1 Cerebellum Midbrain Pons Medulla + + + + ++ + + + + + + ++ +++ ++ +++ +++ +++ + + ++ + + - Thy1-GCaMP6f ++ ++ ++ ++ ++ +++ + + ++ + + + - ++ ++ ++ + ++ +++ + ++ + ++ + 012735 R26 :: CAG ArchT / EGFP Ai40D 021188 R26 :: CAG ChETA/ChR2*H134R ; tdT 2xChETA-tdT • Ca2+ binding (neuron activation) EGFP fluorescence (~510 nm) • Optimized dynamic range, baseline fluorescence, sensitivity & function 017455 R26 :: CAG ChR2*H134R / EYFP Ai32 024109 GCaMP6s : ultrasensitive with slower decay & response kinetics GCaMP6f : fast response kinetics R26 :: CAG hChR2*H134R / tdT Ai27D 012567 R26 :: CAG ReaChR / mCitrine Rosa26 CAG-LSL-ReaChR-mCit 026294 R26 :: CAG eNpHR3.0 / EYFP Ai39 014539 R26 :: CAG GCaMP3 Ai38 014538 RNA pol II :: CAG GCaMP5G PC::G5-tdT 024477 R26 :: CAG GCaMP6f [membrane] R26-Lck-GCaMP6f flox 029626 R26 :: CAG GCaMP6f Ai95D (C57BL/6J) 028865 R26 :: CAG GCaMP6s Ai96 (C57BL/6J) 028866 R26 :: CAG mOrange2 ; EGFP ; mKate2 RGBow reporter 028583 R26 :: CAG Synaptophysin / tdT Ai34D 012570 R26 :: CAG rtTA3 ; mKate2 Rosa26-CAGs-LSL-RIK 029633 CaViAr GCaMP6f::QuasAr2-mOr2 FEATURED IMAGE: Ai95D x ALDH1L1- Allows simultaneous, all-optical electrophysiology CreERT2 double mutant hippocampus showing ALDH1L1 is pan-astrocytic (JAX#s 024105 x 029655). Image courtesy of Drs. Baljit Khakh & Rahul Srinivasan Allows simultaneous Ca2+ and voltage imaging Cre:Tet-Inducible Lines CheRiff : sensitive blue light-activated ChR variant; fused to eGFP Following Cre recombination, mice have dox-inducible / reversible expression of opsins, opsin/xFPs and calcium and glutamate sensors PROM EFFECTOR TIGRE locus (Igs7) ~21.54 Mbp on Chr.9 EXPRESSION NAME R26 :: CAG CheRiff-eGFP:: QuasAr2-mOrange2 Cre-inducible Floxopatch, Optopatch2 028678 TIGRE :: TRE CheRiff-eGFP:: QuasAr2-mOrange2 Cre-inducible & Tet-control Optopatch2 Ai141 029677 TIGRE :: TRE ; CaMK2a TIGRE :: TRE TIGRE :: TRE ; R26 CheRiff-eGFP:: QuasAr3-mCitrine Specific Promoters drive expression of opsin/xFPs, Ca+ sensors and photoactivatable GFP PROMOTER ++ ++ + ++ ++ +++ + + ++ + - ++ ++ + + ++ ++ + + ++ + + - + + ++ + ++ ++ ++ + ++ + + + ++ +++ + +++ +++ +++ + + +++ + ++ + choline acetyltransferase vesicular GABA transporter tryptophan hydroxylase 2 olfactory receptor 160 thymus cell antigen 1 EFFECTOR EXPRESSION ChR2 / EYFP widespread brain ChR2 / EYFP widespread brain mhChR2 / EYFP mhChR2 / EYFP mhChR2 / EYFP PV+ GABAergic interneurons cholinergic neurons GABAergic interneurons COMMON NAME Thy1-ChR2-EYFP line 18 Thy1-ChR2-EYFP line 9 PV-hChR2(H134R)-EYFP line 15 ChAT-ChR2(H134R)-EYFP line 6 VGAT-ChR2(H134R)-EYFP JAX# STOP loxP loxP EFFECTOR Cre-inducible & Tet-control Optopatch3 Ai155 029679 Cre-inducible & Tet-control CaViAr Ai142 029678 PROMOTER EFFECTOR COMMON NAME TIGRE :: TRE TIGRE :: TRE ; R26 GCaMP6f GCaMP6f + tTA GCaMP6f + tTA GCaMP6s GCaMP6fs + tTA GCaMP6fs + tTA iGluSnFr Jaws / EGFP (Halo57/ EGFP) ShChR (Chronos) Ai93D Ai93D;ROSA26-ZtTA Ai93D;CaMK2a-tTA Ai94D Ai94D;ROSA26-ZtTA Ai94D;CaMK2a-tTA Ai85D Ai79D Ai90D TIGRE :: TRE ; CaMK2a TIGRE :: TRE TIGRE :: TRE TIGRE :: TRE JAX# 024103 024107 024108 024104 024112 024115 026260 023529 024100 Cre:FLP Dual Inducible Lines Multiple STOP cassettes prevent transcription of opsin/xFPs or fluorescent protein. PROMOTER STOP loxP STOP loxP EFFECTOR or xFP PROMOTER EFFECTOR STOPS Gly/Glu Chloride Channels R26 :: CAG CatCh / EYFP (ChR2L132C / EYFP) Induce hyperpolarizing Cl current via ivermectin (IVM). GlyCl: glycine-insensitive GlyClαF207A/A288G receptor GluCl: glutamate-insensitive GluClα/βY182F receptor R26 :: CAG ReaChR / mCitrine R26 :: CAG tdT R26 :: CAG tdT ; EGFP R26 :: CAG mCherry ; EGFP frt-STOP-frt :: lox-STOP-lox frt-STOP-frt :: lox-STOP-lox frt-STOP-frt :: lox-STOP-lox frt-STOP-frt :: lox-tdT-lox frt-STOP-frt :: lox-mCherry-lox FEATURED IMAGES: TRE-GlyCl in forebrain (via CaMKII-tTA) or entorhinal cortex (via Klk8-tTA). See JAX#s 029301 and 007004. Images courtesy of Dr. Joanna Jankowsky. COMMON NAME JAX# Ai80D 025109 Rosa26 CAG-FSF024846 LSL-ReaChR-mCit Ai65D 021875 RC::FLTG 026932 RC::FrePe 029486 007612 PROMOTER EFFECTOR EXPRESSION NAME JAX# 007615 TRE hM3Dq Tet- and CNO-inducible Gq TRE-hM3Dq 014093 012355 R26 :: CAG hM3Dq R26-LSL-GqDREADD 026220 014546 R26 :: CAG hM3Dq 014548 R26 :: CAG hM3Dq Cre-inducible mCitrine ; then CNO-inducible Gq FLP-inducible eGFP,; then Cre-inducible mCherry & CNO-inducible Gq Cre-inducible mCherry ; then CNO-inducible Gq insulin 2 hM3Dq CNO-inducible Gq in pancreatic β-cells β-rhM3Dq Tg 028964 TRE hM4Di Tet- and CNO-inducible Gi TRE-hM4Di 024114 R26 :: CAG hM4Di 026219 R26 :: CAG hM4Di Cre-inducible mCitrine ; then CNO-inducible Gi FLP-inducible mCherry ; then Cre- & CNO-inducible Gi R26-LSL-GiDREADD FEATURED IMAGES: Fluorescently-labeled Pet1 serotonergic neurons in adult RC::FPDi 029040 insulin 2 rM3Ds CNO-inducible Gs in pancreatic β-cells β-GsD Tg 028966 GlyClαF207A/A288G Tet- & IVM-inducible GlyCl channel :: eYFP Cre- & IVM-inducible GluCl channel :: eYFP TRE-GlyCl 029301 lower brainstem of RC::FrePe (JAX#029486) x Pet1::Flpe x Egr2::cre (JAX#025744) mice (left, coronal ; right, sagittal). mCherry+ cells are Pet1 serotonergic neurons derived from rhombomere6 or caudal to r6. Intersectional (eGFP+) cells are Pet1 serotonergic neurons from transcription factor Egr2-expressing cell lineage - which derive from rhombomere5 (embryonic hindbrain). Signal enhanced via immunostaining. Images courtesy of Drs. Susan Dymecki and Rachael Brust. R26-LSLGluCleYFP 029691 TPH2-ChR2(H134R)-EYFP line 5 014555 021206 mhChR2 / EYFP serotonergic neurons ChR2*H134R / EYFP M72+ olfactory sensory neurons M72-IRES-ChR2-YFP widespread brain Thy1-GCaMP3 line 6 (C57BL/6) 029860 GCaMP3 TREtight JAX# “Designer Receptors Exclusively Activated by Designer Drugs” are mutant G protein-coupled receptors activated by CNO. hM3Dq: activation Gq-DREADD (burst firing) hM4Di: inhibitory Gi-DREADD (neuronal silencing) rM3Ds: activation Gs-DREADD (increase cAMP). parvalbumin GP4.3 GP4.6 GP4.12 GP5.1 GP5.3 GP5.5 GP5.11 GP5.12 GP5.17 024275 028278 025776 028279 028280 024276 024339 028281 025393 + ++ + + ++ +++ + ++ + + + Ai35D DREADD thymus cell antigen 1 thymus cell antigen 1 Founder line-specific expression of GCaMP6 variants in brain from Dana et al. 2014 PLoS One. 9(9):e108697 Table 1. Line JAX# Arch / GFP QuasAr2-mOrange2:: TIGRE :: TRE GCaMP6f [membrane] 025406 Thy1-GCaMP6 Lines Thy1-GCaMP6s R26 :: CAG CHEMOGENETICS EXPRESSION smooth muscle (incl. vessels) JAX# TIGRE :: TRE Images from Karl Deisseroth Optogenetics website, Williams and Deisseroth 2013 PNAS 110:16287 and Häusser 2014 Nat Methods 11:1012 EFFECTOR loxP COMMON NAME QuasAr3: improved in vivo trafficking; fused to membrane-targeted Citrine D. APPLICATIONS Fiber optic electrode delivers specific wavelengths of light = opens the ion channel = light controls neuronal signaling STOP EFFECTOR Halorhodopsin Archaerhodopsin-3 Channelrhodopsin-2 (NpHR) (Arch) (ChR2) inward chloride ion pump outward proton pump cation channel hyperpolarizes celll hyperpolarizes cell depolarizes cell prevents action causes action potentials prevents action potentials potentials in response to in response to in response to yellow light yellow-green light blue light. C. DESIGN FOR GENETICALLY-ENCODED OPTOGENETIC MODELS Create a targeted mutation or transgene in mouse. Often, a fluorescent protein is engineered to be co-expressed. loxP PROMOTER OptoPatch2 / OptoPatch3 CheRiff-eGFP::QuasAr-mxFP “Cornell / National Heart Lung Blood Resource for Optogenetic Mouse Signaling” genetically encoded calcium indicators and optogenetic effectors - allow combinatorial sensor expression with discrete emission wavelengths in interacting lineages, as well as optically compatible effector/detector pairs. α-SMA PROMOTER Voltage Indicators : OptoPatch and CaViAr CHROMus EFFECTOR Calcium Indicators : GCaMP A floxed-STOP cassette prevents transcription of opsin/xFPs, calcium sensors, photoactivatable GFP, etc. QuasAr2: near infrared-activated, Arch-derived, enhanced voltage indicator; fused to a membrane-targeted dark Orange2 The Jackson Laboratory Resources for Optogenetics, Cre-dependent Optogenetic Tools and Cre Strains for Neurobiology www.jax.org/optogenetics PROMOTER Cre-Inducible Lines GCaMP illustration adapted from Akerboom et al. 2012 J Neurosci 32:13819 Search The Jackson Laboratory Repository / JAX® Mice Database www.jax.org/mouse-search TRANSIENT-SENSORS TRE GCaMP6s Tet-inducible TRE-GCaMP6s line G6s2 024742 synaptosomal-assoc. protein 25 GCaMP6s widespread brain Snap25-2A-GCaMP6s-D 025111 TRE human ubiquitin C PA-GFP widespread UBC PA-GFP 022486 R26 :: CAG GluClα/βY182F RC::FL-hM3Dq 026942 RC::L-hM3Dq 026943 www.jax.org www.jax.org