An AAV-GFP virus containing both AAV serotype 2 Capsid and expressing GFP was prepared. GFP expression under the control of cytomegalovirus immediate-early promoter (CMV) (AAV-GFP, 1.0 × 1013 viral particles/ml; Vector Biolabs, Philadelphia, USA) was used as a control. Human NT-3 subcloned into an AAV vector cassette under the control of the CMV and containing a polyadenylation signal from human B-globin gene was prepared by the Smith Lab (Temple University, Philadelphia, USA; 1.0 × 1012 viral particles/ml).
Aav gfp
Manufactured by Vector Biolabs
Sourced in United States
AAV-GFP is a recombinant adeno-associated virus (AAV) vector that expresses the green fluorescent protein (GFP) gene. It is designed for the transduction and expression of the GFP gene in target cells.
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Lab products found in correlation
Anti inos antibody, by BD (2 mentions)
Aav dj ef1a dio tatalox dse eyfp shinos aav, by Vector Biolabs (2 mentions)
Syringe pump, by Harvard Apparatus (2 mentions)
Live dead cytotoxicity assay, by Thermo Fisher Scientific (1 mentions)
Ump3 microsyringe pump, by World Precision Instruments (1 mentions)
Aav cre gfp, by Vector Biolabs (1 mentions)
Aav2 cre gfp, by Vector Biolabs (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
9 protocols using aav gfp
1
Recombinant AAV-GFP and AAV-NT3 Production
2
Zfp212-KO Mice Stereogenic Injection
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Three-week-old Zfp212-KO mice were used for stereogenic injection. AAV-GFP and AAV-ZNF212 viruses were purchased from Vector Biolabs (USA). Anesthetized mice with pentobarbital (50 mg/kg, intraperitoneal injection) were injected in two positions in the Cb at the following coordinates: DV: − 1.5 AP: − 5, ML: − 1.5/1.5. After 6 weeks, the mice were subjected to behavioral tests and biochemical experiments.
3
Retinal Ganglion Cell Isolation and Transduction
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RGCs were isolated from retinas of postnatal day 6 neonatal mice using the Miltenyi Biotec magnetic cell sorting (MACS) system following a published protocol (Huang et al., 2003 (link); Jiao et al., 2005 (link)). Immediately after isolation, cells were incubated with adeno-associated virus (AAV)-GFP or AAV-p58IPK (Vector Biolabs, Malvern, PA, USA) at 10∧12 GC/ml following standard procedures approved by the Institutional Biosafety Committee at the University at Buffalo. After 24 h, transduced cells were treated with 1 μg/ml tunicamycin (TM) or a vehicle control (0.05% DMSO) for an additional 16 h. Cell viability was examined using the live/dead cytotoxicity assay (Molecular Probes) following the manufacturer’s protocol. Images were analyzed for live cells (green) and dead cells (red), blind to treatment or genotype (see Supplementary Material ).
4
Bicistronic AAV Encoding Secretory CNTF
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The bicistronic adeno-associated viral vector (AAV) encoding and expressing a secretory form of CNTF coupled to mCHERRY (AAV-CNTFmCherry) or vector-alone control linked to green fluorescent protein (AAV-GFP) were made by Vector Biolabs, USA. AAV vectors consisted of an AAV2 DNA backbone in an AAV1 capsid. This particular serotype has been shown to provide excellent transduction of cortical neurons [27 (link)–29 (link)]. Transgene expression was driven by a shortened CAG2 promoter based on the typical cytomegalovirus/chicken beta-actin (CAG) promoter. For bicistronic vectors, the transgene (CNTF) and reporter (mCherry) were linked by a 2A viral peptide sequence, which causes a “translational skip” and results in a 1 : 1 expression of transgene and reporter proteins. The CNTF transgene is a mouse CNTF gene preceded by the secretory signal sequence from mouse pre-pro-NGF, to allow for local secretion of CNTF [30 (link)] (a gift from Prof. M. Sendtner, University of Wurzburg, Germany).
5
Selective Deletion of Bdnf in Dentate Gyrus
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For the deletion of the Bdnf gene selectively in the dentate gyrus, anesthetized adult Bdnfflox/flox mice underwent bilateral stereotaxic injections of AAV-Cre-GFP or AAV-GFP (0.5 μl /side; Vector Biolabs, Malvern, PA) into the dentate gyrus (coordinates: AP = −2.1 mm, ML = ±1.5 mm, DV = −2.3 mm from Bregma), at a rate of 0.1 μL/min with a 33-gauge stainless steel injector connected to a UMP3 micro syringe pump (World Precision Instruments, Sarasota, FL). Additional 5 min were allowed for diffusion and prevention of backflow. Behavioral experiments were conducted 21 days after AAV injection. For intra-dentate gyrus microinjection of inhibitors of AKT and p300 HAT, the mice were anesthetized with 4% chloral hydrate (400 mg/kg, i.p.). AKTi and C646 (0.5 μl/side) were injected bilaterally into the dentate gyrus of adult C57BL/6J mice at a rate of 0.5 μl/min with a 33-gauge stainless steel injector. Additional 5 min were allowed for diffusion and prevention of backflow, and 30 min later, mice were injected with leptin (5 mg/kg, i.p.).
6
Cortical KCC2 Knockdown Model
7
Isolation and Characterization of Astrocytes
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Hypothalami were dissected from post-natal day 1 (P1) pups from Ptcf/f;R26R–YFP mice and maintained in cold DMEM/F-12 (#31330.038, Invitrogen). Tissues were crushed through a 20-μm Nylon mesh (Buisine, Clermont de l’Oise, France) using a cell scraper and cells centrifuged and seeded in T25 flasks in DMEM/F-12 supplemented with 10% heat-inactivated fetal calf serum (FCS) and 100 units/ml of penicillin-streptomycin both from Invitrogen. The cells were incubated at 37 °C under 5% CO2 until confluency (12–15 days in vitro (DIV)) with the medium changed every 3 days. To remove contaminants, the sealed flask was shaken for 24 h at room temperature with one change of equilibrated culture medium. The next day, the cells were split for future use. Astrocytes from Ptcf/f;R26R–YFP mice (DIV 8–18) were seeded in poly-d -lysine (PDL)-coated (Sigma) 24-well plates at a density of 50,000 cells per well. The next day, they were infected with AAV-Cre (#7012) or AAV-GFP (#7006) (Vector Biolabs) MOI 50,000 in 250 μl of serum-free DMEM-F12 + 0.4% AdenoBOOST (#SB-P-AV-101-01, Sirion Biotech) for 6 h. The cells were washed twice in PBS and then cultured in regular medium for 5 days. The medium was changed to serum-free media for 24 h and [3H]-2-deoxy-d -glucose uptake assays, RNA extraction, and immunocytochemistry were performed the next day.
8
Cre-Dependent iNOS Knockdown in Arcuate Nucleus
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Small hairpin RNA (shRNA) targeting murine NOS2, which encodes iNOS,
was cloned into the pAAV-EF1a-DIO-TATAlox-EYFP-U6 vector in which iNOS shRNA
was designed to be expressed in a Cre-dependent manner (Figure 4A ). Before generating AAV, successful iNOS
knockdown was tested in RAW264.7 cells by cotransfecting
pAAV-EF1a-DIO-TATAlox-DSE-EYFP-shiNOS and Cre-recombinase AAV. Cells were
harvested 72 h after transfection and following 24 h-palmitate treatment
(500 μM) (Figure 4B ). iNOS
expression was determined by western blotting using anti-iNOS antibody (BD
Biosciences, #610328). AAV-DJ-EF1a-DIO-TATAlox-DSE-EYFP-shiNOS-AAV was
produced by Vector Biolabs (Malvem, PA). The shiNOS-AAV (3.4 3
109 genome copies in 400 nl) was microinjected bilaterally
into the ARC (6.1 mm deep, 1.6 mm caudal to bregma, 0.1 mm lateral from the
sagittal suture) of LysMtdT or LysMGFP mice under
anesthesia via a syringe pump (Harvard Apparatus, Holliston, MA) at a rate
of 40 nl/min for 10 min (400 nl/injection site). Control animals were
injected with the same amount of GFP-AAV (Vector Biolabs). Successful
injection of AAV was verified by YFP expression or suppressed iNOS
expression in ARC LysM+ cells (Figures 4C and 4D ). Results from animals with successful
injections were included in the data analyses. The experiments were repeated
twice.
was cloned into the pAAV-EF1a-DIO-TATAlox-EYFP-U6 vector in which iNOS shRNA
was designed to be expressed in a Cre-dependent manner (
knockdown was tested in RAW264.7 cells by cotransfecting
pAAV-EF1a-DIO-TATAlox-DSE-EYFP-shiNOS and Cre-recombinase AAV. Cells were
harvested 72 h after transfection and following 24 h-palmitate treatment
(500 μM) (
expression was determined by western blotting using anti-iNOS antibody (BD
Biosciences, #610328). AAV-DJ-EF1a-DIO-TATAlox-DSE-EYFP-shiNOS-AAV was
produced by Vector Biolabs (Malvem, PA). The shiNOS-AAV (3.4 3
109 genome copies in 400 nl) was microinjected bilaterally
into the ARC (6.1 mm deep, 1.6 mm caudal to bregma, 0.1 mm lateral from the
sagittal suture) of LysMtdT or LysMGFP mice under
anesthesia via a syringe pump (Harvard Apparatus, Holliston, MA) at a rate
of 40 nl/min for 10 min (400 nl/injection site). Control animals were
injected with the same amount of GFP-AAV (Vector Biolabs). Successful
injection of AAV was verified by YFP expression or suppressed iNOS
expression in ARC LysM+ cells (
injections were included in the data analyses. The experiments were repeated
twice.
9
Cre-Dependent iNOS Knockdown in Arcuate Nucleus
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