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Paav hsyn dio mcherry

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The PAAV-hSyn-DIO-mCherry is a plasmid vector that contains the hSyn promoter and a double-inverted open reading frame (DIO) cassette, allowing for Cre-dependent expression of the mCherry fluorescent protein. This plasmid is commonly used for neuronal-specific gene expression studies.

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Paav hsyn dio hm3d gq mcherry, by Addgene (7 mentions) Paav hsyn dio hm4d gi mcherry, by Addgene (7 mentions) Clozapine n oxide, by Bio-Techne (2 mentions) Paav ef1a dio mcherry, by Addgene (2 mentions) Paav hsyn dio egfp, by Addgene (2 mentions) Paav ef1a fdio cre, by Addgene (2 mentions) Paav syn flex gcamp6f wpre sv40, by Addgene (2 mentions) Aav ef1a dio gcamp6f p2a nls tdtomato, by Addgene (2 mentions) Paav hsyn1 sio stgtacr2 fusionred, by Addgene (2 mentions) Paav ef1a dio eyfp, by Addgene (1 mentions) Aav1 cag flex gfp wpre, by Addgene (1 mentions) Aav1 cag flex archt gfp, by Addgene (1 mentions) Paav flex tdtomato, by Addgene (1 mentions) Plasmid maxiprep kit, by Qiagen (1 mentions) Aav purification mega kit, by Cell Biolabs (1 mentions)

16 protocols using paav hsyn dio mcherry

1

Chemogenetic Manipulation and Calcium Imaging in Mice

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Clozapine N-oxide (CNO, Tocris) was prepared in dimethyl sulfoxide (DMSO) at concentration of 20 mg/ml and intraperitoneally injected at 5 mg/kg in mice 1 hr. before fear conditioning test. pENN.AAV.CamKII.GCaMP6f.WPRE.SV40 (Addgene 100834-AAV1) was injected to CA1 area 3–4 weeks before lens implantation. The DREADD viruses pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene: 44361-AAV1), pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene: 44362-AAV1), or control virus pAAV-hSyn-DIO-mCherry (50459-AAV1) were injected in the dorsal CA1 to express hM3D, hM4D, or mCherry only, respectively. AAV1.CAG.LSL.tdTomato.bGH (Penn Vector Core: AV-1-ALL856) conditionally expressing tdTomato was injected to CA1 to label SOM cells for slice recording.
He L., Caudill M.S., Jing J., Wang W., Sun Y., Tang J., Jiang X, & Zoghbi H.Y. (2022). A weakened recurrent circuit in the hippocampus of Rett syndrome mice disrupts long-term memory representations. Neuron, 110(10), 1689-1699.e6.
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2

Cre-dependent hDlx AAV virus cloning

Cited 1 time
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For cloning of Cre-dependent hDlx AAV virus, we amplified the hM3D-mCherry and mCherry coding sequences from the plasmid pAAV-hSyn-DIO-hM3D(Gq)-mCherry (a gift from Bryan Roth, Addgene # 44361)57 (link) and pAAV-hSyn-DIO-mCherry (also a gift from Bryan Roth, Addgene # 50459) and cloned them into a pAAV-hDlx-Flex-GFP vector backbone (a gift from Gordon Fishell, Addgene # 83895)58 (link) at AccI and NheI cloning sites. The coding sequence of adra1A was synthesized from Bio Basic Inc. and cloned into a pAAV-hDlx-Flex backbone. The virus was further packaged by Vigene Biosciences Company in AAVdj serotype. pAAV-EF1a-DIO-ChR2-mCherry plasmid (a gift from Karl Deisseroth, addgene # 20297) was purchased from addgene and packaged into AAVs from Vigene Biosciences Company in serotype9. All hDlx AAV viruses were diluted to the range of 1011 to 1012 viral genome per ml with virus dilution buffer containing 350 mM NaCl and 5% D-Sorbitol in PBS.
Fu X., Teboul E., Weiss G.L., Antonoudiou P., Borkar C.D., Fadok J.P., Maguire J, & Tasker J.G. (2022). Gq neuromodulation of BLA parvalbumin interneurons induces burst firing and mediates fear-associated network and behavioral state transition in mice. Nature Communications, 13, 1290.
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3

CRISPR-Mediated Gene Silencing in Neurobiology

Cited 2 times
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The following Adeno-Associated Viruses were used: CRISPR AAVs expressing saCas9 and gRNA against Ucn1, CCK and CART were obtained from GeneCopoeia (MA, USA). 3 AAVs expressing different gRNAs were obtained for each gene. Catalogue numbers are AA01-MCP001598-AD01-3-200 a, b and c (UCN1, both variants GenBank:NM_021290.2 & GenBank:NM_001346010.1), 217AA01-CCPCTR01-AD01-100 (scrambled), 217AA01-MCP291368-AD01-3-200 a, b and c (CCK, GenBank:NM_031161.4) and 217AA01-MCP299483-AD01-3-200 (Cartpt, GenBank:NM_013732.7). AAV1-flex-taCasp3-TEVp (Yang et al., 2013 (link)) DNA was obtained from Addgene (ref. 45580) and produced at the Paris Brain Institute viral core facility. pAAV-EF1a-DIO-ChrimsonR-mRuby2-KV2.1-WPRE-SV40 (Chettih and Harvey, 2019 (link)) AAV9 particules were obtained from Addgene (ref 124603-AAV9). pAAV-Ef1a-DIO-EYFP AAV2 particules were obtained from Addgene (ref 27056-AAV2). pGP-AAV-syn-FLEX-jGCaMP7s-WPRE (AAV1) (Dana et al., 2019 (link)) AAV1 particules were obtained from Addgene (ref 104491-AAV1). pAAV-hSyn-DIO-hM3D(Gq)-mCherry, pAAV-hSyn-DIO-mCherry and pAAV-hSyn-DIO-hM4D(Gi)-mCherry AAV2 particules (Krashes et al., 2011 (link)) were obtained from Addgene (ref 44361-AAV2, 50459-AAV2 and 44362-AAV2).
Topilko T., Diaz S.L., Pacheco C.M., Verny F., Rousseau C.V., Kirst C., Deleuze C., Gaspar P, & Renier N. (2022). Edinger-Westphal peptidergic neurons enable maternal preparatory nesting. Neuron, 110(8), 1385-1399.e8.
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4

Viral Vector Toolkit for Optogenetics and Chemogenetics

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AAV2/1-pEF1a-DIO-hChR2-eYFP (1.82 x 1013 GC/ml, UPenn vector core), AAV1-CAG-FLEX-GFP-WPRE (2 x 1013 GC/ml, UPenn vector core, Addgene 51502), AAVretro-Cre (1.5 x 1014 GC/ml, Vigene)51 , AAV1-CAG-FLEX-ArchT-GFP (4 x 1012 GC/ml, UNC vector core), pAAV-hSyn-DIO-hM3D(Gq)-mCherry (1.3 x 1013 GC/ml, Addgene 44361), pAAV-hSyn-DIO-hM4D(Gi)-mCherry (3 x 1013 GC/ml, Addgene 44362), pAAV-hSyn-DIO-mCherry (4.8 x 1013 GC/ml, Addgene 50459), AAV1-Syn-FLEX-GCamp6s-WPRE-SV4 (Addgene 100845), AAV1-DIO-FLPo-WPRE-hGHpA (1.53 x 1014 GC/ml, Addgene 87306), AAV8-CAG-fDIO-TVA-mCherry (1.1 x 1013 GC/ml, Salk Institute), AAVDJ-CAG-fDIO-oG-WPRE(4.4 x 1013 GC/ml, Salk Institute), EnvA-G-deleted Rabies-GFP (8.13 x 109 GC/ml, Salk Institute) were used in this study. The volume for each injection was 50 nl.
Zhang G.W., Shen L., Tao C., Jung A.H., Peng B., Li Z., Zhang L.I, & Tao H.W. (2021). Medial Preoptic Area Antagonistically Mediates Stress-induced Anxiety and Parental Behavior. Nature neuroscience, 24(4), 516-528.
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5

Dual-promoter AAV Vectors for Cre-dependent EGFP Expression

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The AAV-hSyn-DIO-EGFP-L10a-WPRE-hGH and AAV-Ef1a-DIO-EGFP-L10a-WPRE-hGH plasmids were assembled from pAAV-hSyn-DIO-mCherry (Addgene plasmid #50459) and pAAV-EF1A-DIO-mCherry (Addgene plasmid #50462), which were gifts from Dr. Bryan Roth, and an EGFP-L10a construct, which was a gift from Dr. Anne Schaefer. AAV serotype 5 viruses were prepared by the University of Pennsylvania Vector Core with a titer of 5.97 × 1012 for AAV5-hSyn-DIO-EGFP-L10a-WPRE-hGH and 3.22 × 1012 for AAV5-Ef1a-DIO-EGFP-L10a-WPRE-hGH.
Benthall K.N., Cording K.R., Agopyan-Miu A.H., Wong C.D., Chen E.Y, & Bateup H.S. (2021). Loss of Tsc1 from striatal direct pathway neurons impairs endocannabinoid-LTD and enhances motor routine learning. Cell reports, 36(6), 109511.
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6

Viral Expression of hM4D(Gi) in Cerebellar Lobules

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Adeno-associated viruses (AAV) pAAV-hSyn-DIO-hM4D(Gi)-mCherry and pAAV-hSyn-DIO-mCherry were purchased from Addgene (Watertown, USA). nNos-Cre homozygous mice (~P24–P90) were anesthetized with isoflurane and placed in a stereotaxic frame (Neurostar). For viral injections, a glass electrode filled with AAV virus was placed into the target area according to the corresponding coordinates: Lobules V/VI inclusive (lambda 15–20 mm posterior, 0 mm M/L, 2 mm ventral) and Lobules V/VI exclusive (Lambda ± ~35 mm posterior, ±0 mm M/L, 2 mm ventral). The scalp incision was stapled, and animals were allowed at least 2 weeks to recover and express the virus before FC training (tone + shock in context A) and memory retention tests (tone alone in context B, conducted 24 h after FC). Freezing responses to tones were quantified as described above. Correct location of the virus injection was confirmed postmortem by mCherry fluorescence.
Cariou S., Donovan J.C., Flanagan W.M., Milic A., Bhattacharya N, & Slingerland J.M. (2000). Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America, 97(16), 9042-9046.
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7

Diverse Viral Tools for Neuroscience

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The rAAV5-Ef1a-DIO-hChR2-eYFP, AAV9-CAG-Flex-GFP, and AAV8-hSyn-DIO-KORD-IRES-mCitrine were produced by the University of North Carolina vector core facility (Chapel Hill, North Carolina, USA). The pAAV-Syn-Flex-GCaMP6f-WPRE-SV40 (Catalog number 100833), AAV-Ef1a-DIO-GCaMP6f-P2A-nls-tdTomato (retrograde AAV; Catalog number 51083), pAAV-EF1a-fDIO-Cre (retrograde AAV; Catalog number 121675-AAVrg), pAAV_hSyn1-SIO-stGtACR2-FusionRed (Catalog number 105677-AAV1), pAAV-hSyn-DIO-EGFP (retrograde AAV; Catalog number 50457-AAVrg) and pAAV-hSyn-DIO-mCherry (retrograde AAV; Catalog number 50459-AAVrg) were produced by Addgene (Watertown, MA, USA). The CAV-Flex-FlpO was produced by Montpellier vector platform (Plateforme de Vectorologie de Montpellier (PVM), Biocampus Montpellier, Montpellier, France). The AAVDJ-CAG-fDIO-oG-WPRE-SV40pA was produced by the Viral Vector Core Facility at Salk Institute. The AAV2/8-Ef1a-fDIO-TVA-mCherry was produced by Z. Josh Huang’s lab at CSHL. The Rbv-CVS-N2c-dG-GFP (the optimized rabies virus)36 (link) was produced by HHMI Janelia Research Campus. The AAV8-CaMKIIa-FDO-GCaMP6f (Flp-off) virus was produced by K. Deisseroth’s lab at Stanford University. All viral vectors were aliquoted and stored at –80 °C until use.
Zhang X., Guan W., Yang T., Furlan A., Xiao X., Yu K., An X., Galbavy W., Ramakrishnan C., Deisseroth K., Ritola K., Hantman A., He M., Huang Z.J, & Li B. (2021). Genetically identified amygdala-striatal circuits for valence-specific behaviors. Nature neuroscience, 24(11), 1586-1600.
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8

Cre-dependent DREADD expression with AAV

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pAAV‐hSyn‐DIO‐hM3D(Gq)‐mCherry (#44361; Addgene, MA, USA) and pAAV‐hSyn‐DIO‐mCherry (#50459; Addgene) were used for Cre‐dependent expression of an excitatory DREADD hM3Dq. Adeno‐associated virus (AAV) was packaged in the KIST Virus Facility (Seoul, Republic of Korea). Virus titer was 9.86 * 1012 GC/ml (Genome Copy/ml) for pAAV‐hSyn‐DIO‐mCherry and 3.70 * 1012 GC/ml for pAAV‐hSyn‐DIO‐hM3D(Gq)‐mCherry.
Kim B, & Im H. (2020). Chronic nicotine impairs sparse motor learning via striatal fast‐spiking parvalbumin interneurons. Addiction Biology, 26(3), e12956.
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9

Diverse AAV Virus Production for Neuroscience

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The following AAV (adeno-associated virus) viruses were produced at the Gene Therapy Center Vector Core at the University of North Carolina Chapel Hill: AAV-cFos-hChR2(H134R)-EYFP-Pest-no-WPRE, AAV-hSyn-EYFP, AAV8-hSyn-DIO-hM3D(Gq)-mCherry, and pAAV-EF1a-DIO-mCherry (UNC Vector Core, USA). The AAV5-Syn.Flex.GCaMP6s virus was obtained from the Penn Vector Core (USA). pAAV-hSyn-DIO-hM4D(Gi)-mCherry, pAAV-hSyn-DIO-mCherry, and pAAV-FLEX-tdTomato (produced with a retrograde serotype AAVrg) were obtained from Addgene (USA).
Peters C., He S., Fermani F., Lim H., Ding W., Mayer C, & Klein R. (2023). Transcriptomics reveals amygdala neuron regulation by fasting and ghrelin thereby promoting feeding. Science Advances, 9(21), eadf6521.
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10

Diverse Viral Tools for Neuroscience

Cited 1 time
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The rAAV5-Ef1a-DIO-hChR2-eYFP, AAV9-CAG-Flex-GFP, and AAV8-hSyn-DIO-KORD-IRES-mCitrine were produced by the University of North Carolina vector core facility (Chapel Hill, North Carolina, USA). The pAAV-Syn-Flex-GCaMP6f-WPRE-SV40 (Catalog number 100833), AAV-Ef1a-DIO-GCaMP6f-P2A-nls-tdTomato (retrograde AAV; Catalog number 51083), pAAV-EF1a-fDIO-Cre (retrograde AAV; Catalog number 121675-AAVrg), pAAV_hSyn1-SIO-stGtACR2-FusionRed (Catalog number 105677-AAV1), pAAV-hSyn-DIO-EGFP (retrograde AAV; Catalog number 50457-AAVrg) and pAAV-hSyn-DIO-mCherry (retrograde AAV; Catalog number 50459-AAVrg) were produced by Addgene (Watertown, MA, USA). The CAV-Flex-FlpO was produced by Montpellier vector platform (Plateforme de Vectorologie de Montpellier (PVM), Biocampus Montpellier, Montpellier, France). The AAVDJ-CAG-fDIO-oG-WPRE-SV40pA was produced by the Viral Vector Core Facility at Salk Institute. The AAV2/8-Ef1a-fDIO-TVA-mCherry was produced by Z. Josh Huang’s lab at CSHL. The Rbv-CVS-N2c-dG-GFP (the optimized rabies virus)36 (link) was produced by HHMI Janelia Research Campus. The AAV8-CaMKIIa-FDO-GCaMP6f (Flp-off) virus was produced by K. Deisseroth’s lab at Stanford University. All viral vectors were aliquoted and stored at –80 °C until use.
Zhang X., Guan W., Yang T., Furlan A., Xiao X., Yu K., An X., Galbavy W., Ramakrishnan C., Deisseroth K., Ritola K., Hantman A., He M., Huang Z.J, & Li B. (2021). Genetically identified amygdala-striatal circuits for valence-specific behaviors. Nature neuroscience, 24(11), 1586-1600.
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