Download R26 :: CAG GCaMP6f - The Jackson Laboratory

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
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Thy1-GCaMP6f
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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
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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
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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