Paav cag tdtomato

Manufactured by Addgene

Sourced in United States

The PAAV-CAG-tdTomato is a plasmid that contains the CAG promoter driving the expression of the tdTomato fluorescent protein. The CAG promoter is a strong, ubiquitous promoter, and the tdTomato protein is a red fluorescent protein that can be used for imaging and tracking purposes.

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10 protocols using paav cag tdtomato

Plasmid Purification and AAV Production

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Plasmids for transfection were purified by using Qiagen's endotoxin-free HiSpeed Gigaprep kit (Qiagen) and quantified by spectrophotometry. The integrity of the cis plasmids and ITRs were confirmed by restriction digest and NGS. Complete reagent information is listed in Supplementary Table S5.
The following plasmids were used to prepare the rAAV described in this study; pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA (Addgene 20298, a gift of Karl Deisseroth, unpublished) AAV-Cre-GFP (Addgene 68544, a gift of Eric Nestler), AAV-EF1a-DIO-GCaMP6s-P2A-nls-dTomato (Addgene 51082, a gift of Jonathan Ting), AAV-FLEX-rev-ChR2(H134R)-mCherry (Addgene 18916, a gift from Scott Sternson), rAAV2-retro helper (Addgene 81070, a gift of Alla Karpova), pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene 44361, a gift of Bryan Roth), pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene 44362, a gift of Bryan Roth), pAAV-hSyn-hM3D(Gq)-mCherry (Addgene 50474, a gift of Bryan Roth, unpublished), pAAV-hSyn-EGFP(Addgene 50465, a gift of Bryan Roth, unpublished), pAAV-CAG-GFP (Addgene 37825, a gift from Edward Boyden, unpublished), pAAV-CAG-tdTomato (Addgene 59462, a gift of Edward Boyden, unpublished), and pUCmini-iCAP-PHP.eB (Addgene 103005, a gift of Viviana Gradinaru).^{15–20 (link)}

Guerin K., Rego M., Bourges D., Ersing I., Haery L., Harten DeMaio K., Sanders E., Tasissa M., Kostman M., Tillgren M., Makana Hanley L., Mueller I., Mitsopoulos A, & Fan M. (2020). A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations from Viral DNA Extracts. Human Gene Therapy, 31(11-12), 664-678.

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Retrograde AAV Vector Expression

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Retrograde Adeno-Associated Viral vectors [AAVrg; i.e., AAV-CAG-hChR2-H134R-tdTomato (Cat#: 28017-AAVrg) and pAAV-CAG-tdTomato (codon diversified) (Cat#: 59462-AAVrg)] that allow the expression of tdTomato (tdTom) and/or the light sensitive cation channel, channelrhodopsin-2 (ChR2), were obtained from Addgene, and used at a titer of ≥7 × 10^12 vg/mL.

Elsaafien K., Harden S.W., Johnson D.N., Kimball A.K., Sheng W., Smith J.A., Scott K.A., Frazier C.J., de Kloet A.D, & Krause E.G. (2022). A Novel Organ-Specific Approach to Selectively Target Sensory Afferents Innervating the Aortic Arch. Frontiers in Physiology, 13, 841078.

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Engineered Barcode Viral Constructs

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Plasmid pAAV-CAG-tdTomato (Addgene, #59462) was first modified by replacing tdTomato and WPRE with EGFP by T4 DNA Ligase mediated ligation. A 15 bp barcode sequence was then inserted after the stop codon of EGFP, linked by EcoRI restriction enzyme recognition site. Sequences barcode 0 representing the AI target, CTGCACCGACGCATT; barcode 1 (DMS target), GAAGGCACAGACTTT; barcode 2 (MD target), GTTGGCTGCAATCCA; barcode 3 (BLA target), AAGACGCCGTCGCAA; barcode 4 (LH target), TATTCGGAGGACGAC. Other barcode sequences used for IHC include barcode 10, AGCTATGCACGATCA; barcode 206, GCGTAAGTCTCCTTG; barcode 210, CCTGTATGCGTGGAG. Engineered viruses were produced by Gene Editing Core Facility, Center for Excellence in Brain Science and Intelligence Technology.

Xu P., Peng J., Yuan T., Chen Z., He H., Wu Z., Li T., Li X., Wang L., Gao L., Yan J., Wei W., Li C.T., Luo Z.G, & Chen Y. (2024). High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling. eLife, 13, e85419.

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AAV-Mediated Cell Manipulation in Mice

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The following constructs were used: pAAV-CAG-tdTomato (Addgene plasmid #59462) was used to generate AAV-PHP.S-CAG-tdTomato virus (Addgene); and pAAV-mCherry-flex-DTA (Addgene plasmid #58536) was used to generate AAV2/PHP.S-mCherry-flex-DTA virus (BCH viral core). For FGF18 overexpression, the coding sequence of Fgf18 (with HA tag) was cloned into the pAAV backbone. The plasmid was further used to generate AAV8-CAG-FGF18–3XHA virus (Welgen).
All AAV viruses were injected intradermally. Viral stock was diluted to a concentration of 1E12 gc/ml with saline (0.9% NaCl). 50 μl of the diluted virus was injected once intradermally. Dorsal skin was collected 6 days following injection of AAV-PHP.S-CAG-tdTomato, 10 days following AAV8-CAG-FGF18–3XHA injection, and 18 days following AAV2/PHP.S-mCherry-flex-DTA. For APM ablation, AAV2/PHP.S-mCherry-flex-DTA was injected as described above into Myh11-CreER mice.

Shwartz Y., Gonzalez-Celeiro M., Chen C.L., Pasolli H.A., Sheu S.H., Fan S.M., Shamsi F., Assaad S., Lin E.T., Zhang B., Tsai P.C., He M., Tseng Y.H., Lin S.J, & Hsu Y.C. (2020). Cell types promoting goosebumps form a niche to regulate hair follicle stem cells. Cell, 182(3), 578-593.e19.

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Viral Construct Design and Packaging

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The CreOn-FlpOff and FlpOn-CreOff ExBoX viral constructs were designed using ApE (https://jorgensen.biology.utah.edu/wayned/ape/) and Vector Builder (https://en.vectorbuilder.com/) free software. The plasmids were synthesized and assembled by Vector Builder. Plasmid DNA was purified using the Qiagen Maxi Prep kit (Qiagen Cat#10023). AAVs were also packaged by Vector Builder into AAV2/9. pENN.AAV.CamKII 0.4. Cre. SV40 (AAV5) (deposited by James M. Wilson; Addgene #105558; RRID:Addgene_105558), pAAV-CAG-TdTomato (AAV5) (deposited by Edward Boyden; Addgene #59462; RRID:Addgene_59462) and pCAG-FLEX-EGFP-WPRE (AAV9) (deposited by Hongkui Zeng; Addgene #51502;; RRID:Addgene_51502) were obtained from Addgene (Oh et al., 2014 (link)). rAAV5/AAV-EF1α-FlpO-WPRE and rAAV9/CAG-GFP were obtained from the UNC GTC Vector Core. pENN.AAV.hSyn.Cre.hGH was Addgene #105555 (RRID:Addgene_105555) deposited by James M. Wilson. All the ExBoX constructs designed for this study, along with the maps and sequences, have been deposited in Addgene. Addgene IDs of ExBoX plasmids can be found in Table S2.

Ubina T., Vahedi-Hunter T., Agnew-Svoboda W., Wong W., Gupta A., Santhakumar V, & Riccomagno M.M. (2021). ExBoX – a simple Boolean exclusion strategy to drive expression in neurons. Journal of Cell Science, 134(20), jcs257212.

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Retrograde Transporter Delivery to Nucleus Accumbens and vBNST

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For histology experiments, C57Bl/6J mice (n=2 mice per sex) were anesthetized with isoflurane in O2 (4% induction and 1% maintenance, 2 L/min) and bilaterally microinjected with 500 nl (100 nL/min) of an AAV containing a green retrograde fluorescent transporter (pAAV-CAG-GFP; Addgene) into the Nucleus Accumbens (+1.6 AP, ± 1.5 ML, −4.4DV, mm relative to bregma, angle 10^o) and 500 nl of a red retrograde fluorescent transporter (pAAV-CAG-tdTomato; Addgene) into to the vBNST (+0.16 AP, ± 0.9 ML, −4.8DV, mm relative to bregma). All subjects were perfused after 1 week.
For electrophysiology experiments, C57Bl/6J mice were anesthetized with isoflurane in O2 and injected with 500nl of pAAV-CAG-GFP into the vBNST for IC-cell body expression. The time from virus injection to the start of slice electrophysiology experiments was 2 weeks for all subjects.

Marino R.A., Girven K.S., Figueiredo A., Navarrete J., Doty C, & Sparta D.R. (2021). Binge ethanol drinking associated with sex-dependent plasticity of neurons in the insula that project to the bed nucleus of the stria terminalis. Neuropharmacology, 196, 108695.

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Engineered AAV Capsid Variants for In Vivo Studies

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The AAV capsid variants such as AAV-MaCPNS1 (Addgene plasmid # 185136) and AAV-MaCPNS2 (Addgene plasmid # 185137) capsids were built by inserting 7-mer peptides between AAs 588–589 of the AAV9 cap gene in the pUCmini-iCAP-PHP.B backbone ((Deverman et al. 2016 (link)), Addgene plasmid # 103002). The AAV-PHP.S capsid was described previously ((Chan et al. 2017 (link)), Addgene plasmid # 103006).
For in vivo validation of AAV capsids, we packaged the vectors with a single-stranded (ss) rAAV genome: pAAV:CAG-2xNLS-EGFP (Deverman et al. 2016 (link)) (available from Caltech CLOVER Center upon request, a similar version with 1xNLS is in Addgene, plasmid # 104061), pAAV:CAG-EGFP, pAAV:hSyn1-tdTomato (a gift from Hongkui Zeng, Addgene plasmid # 51506, (Oh et al. 2014 (link))), pAAV:CAG-tdTomato (a gift from Edward Boyden, Addgene plasmid # 59462), pAAV:hSyn-DIO-hM3D(Gq)-mCherry (a gift from Bryan Roth, Addgene plasmid # 44361, (Krashes et al. 2011 (link))). To make the pAAV:CAG-jGCaMP8s plasmid, jGCaMP8s was synthesized as a gBlocks Gene Fragment (IDT) based on the sequence in the plasmid pGP-AAV-CAG-FLEX-jGCaMP8s-WPRE (Addgene plasmid # 162380) and subcloned into the plasmid pAAV:CAG-EGFP by replacing the EGFP gene.

Chen X., Kumar S.R., Adams C.D., Yang D., Wang T., Wolfe D.A., Arokiaraj C.M., Ngo V., Campos L.J., Griffiths J.A., Ichiki T., Mazmanian S.K., Osborne P.B., Keast J.R., Miller C.T., Fox A.S., Chiu I.M, & Gradinaru V. (2022). Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous systems. Neuron, 110(14), 2242-2257.e6.

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Expressing tdTomato in Cells

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pSuper-venus vector was described previously.^{61 (link)} pAAV-CAG-tdTomato was purchased from Addgene (Cambridge, MA, USA).

Ka M., Kook Y.H., Liao K., Buch S, & Kim W.Y. (2016). Transactivation of TrkB by Sigma-1 receptor mediates cocaine-induced changes in dendritic spine density and morphology in hippocampal and cortical neurons. Cell Death & Disease, 7(10), e2414-.

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Diverse Viral Vectors for Neuroscience Research

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We used the following commercially available viruses: pGP-AAV-syn-FLEX-jGCaMP7s-WPRE (3.1×10¹³ GC/mL, Addgene, 104491-AAV9); pAAV-CaMKIIa-EGFP (2.1×10¹³ GC/mL, Addgene, 50469-AAV8); pAAV-CaMKIIa-hM4D(Gi)-mCherry (2.1×10¹³ GC/mL, Addgene, 50477-AAV8); AAV.CamKII.GCaMP6s.WPRE.SV40 (2.1×10¹³ GC/mL, Addgene, 107790-AAV9); pAAV-CAG-tdTomato (1.3×10¹³ GC/mL, Addgene, 59462-AAVrg); AAV9-hSyn-NE2h (2.37×10¹³ GC/mL, Vigene Biosciences, YL10074-AAV9); pENN.AAV.hSyn.Cre.WPRE.hGH (1.2×10¹³ GC/mL, Addgene, 105553-AAVrg); pAAV.synP.DIO.EGFP.WPRE.hGH (4.3×10¹³ GC/mL, Addgene, 100043-AAV9); pAAV-hSyn-DIO-hM4D(Gi)-mCherry (1.2×10¹³ GC/mL, Addgene, 44362-AAV5).

Corona A., Choe J., Muñoz-Castañeda R., Osten P, & Shea S.D. (2023). A circuit from the locus coeruleus to the anterior cingulate cortex modulates offspring interactions in mice. Cell reports, 42(7), 112771.

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Antibody and Reagent Database for Neuroscience Research

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The following antibodies were used in this study: C-FOS (Cell Signaling, #2250, Clone#9F6, rabbit, 1:500); GFP (Abcam, ab13970, chicken, 1:1000); RAGC (Cell Signaling, #5466, rabbit, 1:500); TIPRL (Abcam, #ab70795, rabbit, 1:500); SHANK2 (Cell Signaling, #12218, rabbit, 1:500); SYNPO (Acris, #BM5086P, rabbit, 0.1 μg/ml); PSD95 (Cell Signaling, #2507, rabbit, 1:1000); HSP90 serum (gift from Dr Chung Wang, IMB, Taiwan, rabbit, 1:2000)^{21 (link)}; p-mTOR (Cell Signaling, #2448, rabbit, 1:500); mTOR (Cell Signaling, #2972S, rabbit, 1:500); S6K (Cell Signaling, #2708S, rabbit, 1:500); p-S6K70 T389 (Cell Signaling, #9205S, rabbit, 1:500); 4EBP (Cell Signaling, #9644S, rabbit, 1:500). The following reagents were obtained commercially: ProHance (Gadolinium contrast agent, Bracco Diagnostics, #111181); Vectastain Elite ABC kit (Vector Labs, #PK-6102); C21 (Tocris, #6422); OCT (Tissue-TeK, 4583); ZnSO₄ (Sigma-Aldrich, #Z0251); 30 ppm zinc diet (Research Diets, D19410B). AAV vectors used in this study were: pAAV-CamKII-hM3D(Gq)-mCherry (Addgene, #50476); pAAV-CamKII-hM4D(Gi)-mCherry (Addgene, #50477)^{22 (link)}; pAAV-CAG-tdTomato (Addgene, #59462).

Yen T.L., Huang T.N., Lin M.H., Hsu T.T., Lu M.H., Shih P.Y., Ellegood J., Lerch J, & Hsueh Y.P. (2022). Sex bias in social deficits, neural circuits and nutrient demand in Cttnbp2 autism models. Brain, 146(6), 2612-2626.

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