All animal procedures were conducted in accordance with U.S. National Institutes of Health guidelines, as approved by the University of Colorado Institutional Animal Care and Use Committee (IACUC). Mice were housed in a 12 light /12 dark cycle at room temperature (∼22 °C), 40–60% relative humidity. For intravitreal virus injections, mice of ages p40–360 of either sex were anaesthetized with isoflurane; ophthalmic proparacaine and phenylephrine were applied for pupil dilation and analgesia. A small incision at the border between the sclera and the cornea was made with a 30 gauge needle. 1 µL of AAV solution was injected with a blunt tip (30 gauge) modified Hamilton syringe (www.borghuisinstruments.com ). AAV9.hsyn.iGluSnFR. WPRE.SV40, (a gift from Loren Looger, Addgene plasmid # 98929 ; RRID:Addgene_98929; 1013 vg/mL in water) was injected into the vitreous humor of wild type mice (C57BL/6J, Jackson laboratory, www.jax.org ). To express iGluSnFR in SACs only, AAV9.hsyn.FLEX.iGluSnFR.WPRE.SV40 (a gift from Loren Looger, Addgene plasmid # 98931 ; RRID:Addgene_98931, similar concentration) was used in ChAT-Cre transgenic mice. AAV9-pGP-AAV-syn-jGCaMP7f-WPRE (Addgene plasmid # 104488 ; RRID:Addgene_104488) was used to measure intracellular calcium levels. Experiments on retinas from all animal groups were performed 2–6 weeks following virus injection.
Wpre sv40
Manufactured by Addgene
Sourced in United States
WPRE.SV40 is a genetic sequence that can be incorporated into viral vectors to enhance gene expression. It contains the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and the Simian Virus 40 (SV40) termination sequence. WPRE can increase the expression of the target gene, while the SV40 sequence provides a transcriptional termination signal.
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15 protocols using wpre sv40
1
Intravitreal Gene Delivery and Imaging
2
Optogenetic Manipulation of Neuronal Activity
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Channelrhodopsin fused with superfolder GFP (ChR2-sfGFP) was designed and synthesized from published sequences using codon optimization for Mus musculus (DNA2.0). To express ChR2-sfGFP in the Pvalb-IRES-Cre mouse, the faithful flexed AAV vector under the control of the human synapsin promoter (aavSyn-Jx) was generated using a PCR-amplified human synapsin promoter and lox66/lox7 sites38 (link). For the Cre-dependent switch “on” version (aavSyn-Jx-rev-ChR2-sfGFP), ChR2-sfGFP was reversely inserted into the aavSyn-Jx via the HindIII and EcoRV restriction-enzyme sites. The viruses were produced with serotype 1 or 7 and purified using cesium chloride gradients39 (link). For optogenetic inhibition, the AAV9.CBA.Flex.Arch-GFP.WPRE.SV40 (Addgene 22222) virus was used. The reason we utilized the Arch virus was that it provides optimal terminal expression and has been shown to inhibit neurotransmitter release40 (link)–42 (link). Although a recent study reported that terminal inhibition using an Arch virus paradoxically enhanced terminal release40 (link), it was only apparent after long-duration illumination; we used short-duration illumination (30 s), which does not significantly increase spontaneous neurotransmitter release as this previous study has shown. The retrograde CAV2-Cre virus was purchased from the Montpellier Vector Platform, France (titer ~2.5 × 10E12 pp/mL).
3
Fluorescent Protein and CRISPR Viral Delivery
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Adeno-associated viruses (AAVs) were used encoding the red fluorescent
protein tdTomato, enhanced yellow fluorescent protein (eYFP) or the
single guide RNA (sgRNA) that cleaves Kv3.4/KCNC4:
pENN.AAV.CAG.tdTomato.WPRE.SV40 (Addgene #105554),
pAAV.CamKII(1.3).eYFP.WPRE.hGH (Addgene #10522),
U6.sgRNA(mKv3.4).CMV.saCas9, respectively. Prior to use, 2 µL of
pENN.AAV.CAG.tdTomato.WPRE.SV40 (1 × 1013 GC/mL) was
diluted with an equal volume of 0.1 M phosphate buffer saline (PBS)
while 2 µL of pAAV.CamKII(1.3).eYFP.WPRE.hGH
(1 × 1013 GC/mL) was mixed with 2 µL of
U6.sgRNA(mKv3.4).CMV.saCas9 (1 × 1011 GC/mL).
protein tdTomato, enhanced yellow fluorescent protein (eYFP) or the
single guide RNA (sgRNA) that cleaves Kv3.4/KCNC4:
pENN.AAV.CAG.tdTomato.WPRE.SV40 (Addgene #105554),
pAAV.CamKII(1.3).eYFP.WPRE.hGH (Addgene #10522),
U6.sgRNA(mKv3.4).CMV.saCas9, respectively. Prior to use, 2 µL of
pENN.AAV.CAG.tdTomato.WPRE.SV40 (1 × 1013 GC/mL) was
diluted with an equal volume of 0.1 M phosphate buffer saline (PBS)
while 2 µL of pAAV.CamKII(1.3).eYFP.WPRE.hGH
(1 × 1013 GC/mL) was mixed with 2 µL of
U6.sgRNA(mKv3.4).CMV.saCas9 (1 × 1011 GC/mL).
4
Viral Tracing of Auditory Cortex Axons
5
GCaMP6s Imaging in Mouse Cortex
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Craniotomy survival surgery, virus injection (AAV1.Syn.Flex.GCaMP6s.WPRE.SV40: Addgene ID: 100845-AAV1), GCaMP6s imaging, motion correction, and cell segmentation were as reported in reference (Mineault et al., 2016 (link)). The coordinates used for these studies in ALM were 2.8mm anterior to Bregma and 1.5mm lateral of the sagittal suture. For the experiments in primary visual cortex, the coordinates used were 1mm anterior of Lambda and 3mm lateral of the sagittal suture.
6
Intraventricular Injection for Stable GCaMP6s Expression
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To induce widespread, rapid and stable expression of the calcium indicator GCaMP6s in hippocampal neurons at early postnatal stages, we intraventricularly injected a viral solution (pAAV.Syn.GCaMP6s.WPRE.SV40, Addgene #100843-AAV1) at P0 (Postnatal day zero) in mouse pups of either sex (Fig. 1A,B ). This injection protocol was adapted from already published methods (Kim et al., 2013 (link), 2014 (link)). Mouse pups were anesthetized on ice for 3–4 min, and 2 µl of the viral solution were injected in the left lateral ventricle which coordinates were estimated at the 2/5 of the imaginary line between the λ and the eye at a depth of 400 µm. Expression of GCaMP was checked on slices and was sufficient for in vivo imaging as early as P5, which is consistent with already published data (Kim et al., 2014 (link)). In addition, GCaMP expression, brightness, and kinetics of the reporter was then stable throughout all developmental stages used (data not shown).
7
Targeted Deletion of Progestogen Receptors in Entorhinal Cortex
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AAV9 expressing GFP-Cre under the control of CamKII promoter (pENN.AAV.CamKII.HI.GFP-Cre.WPRE.SV40, Addgene #105551) was used to delete the expression of PRs in the EC of adult PRfl/fl males. Control mice were injected with AAV9 expressing GFP under the control of CamKII promoter (pENN.AAV.CamKII0.4.eGFP.WPRE.rBG, Addgene # 105541). The injections were performed bilaterally. The stereotaxic coordinates used for viral injections were anteroposterior [AP] − 4.6; mediolateral [ML], −/+ 3.0; dorsoventral [DV], − 3.7 and-3.0 from dura for central and medial EC and AP − 3.7, ML −/+ 4.2, and DV − 3.5 and − 3.0 from dura for lateral EC. A Hamilton Company (Reno, NV) syringe (Hamilton 7000 Glass, 1 μl, 0.3302 mm) was loaded with virus solution and mounted in the peristaltic pump holder (Harvard Apparatus, Holliston, MA; P-1500), 200 nl of the virus was injected at each site at a flow rate of 200 nl/min. The experiments were performed two weeks after viral injection. At the end of the experiment, viral transduction of the EC cortical neurons was confirmed by evaluating GFP expression in the brain slices.
8
Neurotropic AAV-Mediated Ca2+ Imaging in Hippocampal Slices
9
Labeling Thalamo-cortical Projections in Mice
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Two-month-old mice were subjected to unilateral viral injections. To label mediodorsal thalamocortical projections, 0.3–0.35 μl of AAV vectors (AAV9.CAG.hCHR2(H134R)-mCherry.WPRE.SV40; Addgene Cat No 100054-AAV9, titer: 2.92 × 1013 GC/ml) were injected to the mediodorsal thalamus. To label PV interneurons at the dACC, 0.3 μl of AAV vector (AAV-FLEX-rev-ChR2(H134R)-mCherry, Addgene Cat No 18916-AAV9, titer: 1 × 1013 GC/ml) were injected to the dACC. The following stereotaxic coordinates were used: mediodorsal thalamus, − 1.58 mm from Bregma, 0.44 mm lateral from midline, and 3.20 mm vertical from cortical surface; dorsal ACC, 1.42 mm from Bregma, 0.35 mm lateral from midline, and 2.3 mm vertical from cortical surface. To ensure minimal leak into surrounding brain areas, injection pipettes remained in the brain for 5 min after injection before being slowly withdrawn. Electrophysiological studies were carried 2–3 weeks later to allow transgene expression from these viral vectors.
10
BLA Neuronal Activity Imaging in Rats
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Surgery occurred prior to onset of behavioral training. Rats (N = 11) were infused bilaterally with adeno-associated virus (AAV) expressing the genetically encoded calcium indicator GCaMP6f under control of the calcium/calmodulin-dependent protein kinase (CaMKII) promoter (pENN.AAV5.CAMKII.GCaMP6f.WPRE.SV40, Addgene, Watertown, MA) to drive expression preferentially in principal neurons. Virus (0.5 µl) was infused a rate of 0.1 µl/min into the BLA [AP: −2.7 (N = 5) or −3.0 (N = 6); ML:±5.0; DV: −8.6 mm from bregma] using a 28-gauge injector. Injectors were left in place for an additional 10 min to ensure adequate diffusion and to minimize off-target spread along the injector tract. Optical fibers (200 µm diameter, 0.37 numerical aperture (NA), Neurophotometrics, San Diego, CA) were implanted bilaterally 0.2 mm dorsal to the infusion site to allow subsequent imaging of GCaMP fluctuations in BLA neurons. These procedures were replicated in a separate group of subjects (N = 6) that served as unpaired CSØ control. Behavioral training commenced approximately 3–4 weeks after surgery to allow sufficient expression in BLA neurons.
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