Paav2 8

Manufactured by Addgene

PAAV2/8 is a plasmid vector used for adeno-associated virus (AAV) production. It contains the necessary elements for packaging AAV serotypes 2 and 8 viruses.

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Lab products found in correlation

Paddeltaf6, by Addgene (2 mentions) Pucmini icap php s, by Addgene (1 mentions) Nnk degenerate primers, by Integrated DNA Technologies (1 mentions) Paav2 2, by Addgene (1 mentions) Pucmini icap php eb, by Addgene (1 mentions) Paav2 1, by Addgene (1 mentions) Paav2 5, by Addgene (1 mentions) Paav2 9, by Addgene (1 mentions) Px601, by Addgene (1 mentions) Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions) Seamless cloning kit, by Beyotime (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Restriction endonuclease, by New England Biolabs (1 mentions)

4 protocols using paav2 8

Optimized AAV Library Generation

Cited 1 time

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The plasmids used for in vivo selection were adapted from the previous publication^{54 (link)}. rAAV-pUBC-sfGFP-Cap and AAV2/9-REP-AAP was generated from pUBC-mCherry-rAB (Addgene, 115239), pUCmini-iCAP-PHP.S (Addgene, 103006), pAAV2/8 (Addgene, 112864). No in-cis-Lox module or transgenic Cre lines were used given the anatomical separation of DRGs and iWAT. The ROOT capsid library was generated by inserting random heptamers using NNK degenerate primers (Integrated DNA technologies) between the 588 and 589 sites of AAV9 by Gibson assembly as previously described^{54 (link)}.

Wang Y., Leung V.H., Zhang Y., Nudell V.S., Loud M., Servin-Vences M.R., Yang D., Wang K., Moya-Garzon M.D., Li V.L., Long J.Z., Patapoutian A, & Ye L. (2022). The role of somatosensory innervation of adipose tissues. Nature, 609(7927), 569-574.

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Engineered AAV Vectors with Diverse Tropisms

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The single strand AAV (ssAAV) vectors were based on the AAV-hGFAP-GFP vector (40 (link)) by replacing the hGFAP promoter with the hIBA1 promoter. The self-complementary AAV (scAAV) vectors were based on the scAAV-CAG-GFP vector (Addgene #83279) by replacing the CAG promoter with the hIBA1 promoter. All vectors were verified through restriction enzyme digestions and DNA sequencing. AAV viruses were packaged with pAd-deltaF6 (Addgene #112867) and one of the following helper plasmids: pAAV2/1 (Addgene #112862), pAAV2/2 (Addgene #104963), pAAV2/5 (Addgene #104964), pAAV2/6 (Addgene #110660), pAAV2/6m (20 (link)), pAAV2/8 (Addgene #112864), pAAV2/9 (Addgene #112865), or pUCmini-iCAP-PHP.eB (Addgene #103005). Briefly, HEK293T cells were transfected with the packaging plasmids and a vector plasmid. Three days later, virus was collected from the cell lysates and culture media. Virus was purified through iodixanol gradient ultracentrifugation, washed with PBS, and concentrated with 100K PES concentrator (Pierce^™, Thermo Scientific). Viral titers were determined by quantitative PCR with ITR primers (forward: 5-GGAACCCCTAGTGATGGAGTT-3; reverse: 5-CGGCCTCAGTGAGCGA-3).

Serrano C., Cananzi S., Shen T., Wang L.L, & Zhang C.L. (2023). Simple and Highly Specific Targeting of Resident Microglia with Adeno-Associated Virus. bioRxiv.

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AAV8 Viral Particle Production

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To generate AAV8 viral particles, 293T cells in a 15cm dish (1 million cells, culture media: DMEM supplemented with 10%v/v fetal bovine serum) were transfected with pAAV2/8 (Addgene #112864), pAdDeltaF6 (Addgene #112867), and pX601 (Addgene #61591). Seventy-two hours later, media was removed and AAV particles were purified by Iodixanol gradient ultracentrifugation method (Millipore Sigma) according to the manufacturer’s instructions. The viral particles were further concentrated by Amicon^® Ultra-15 centrifugal filter units (Millipore Sigma) according to the manufacturer’s instructions. Viral titers were determined via a PCR based method utilizing the primers described above. The AAV8 viral particles (2.0×10¹² genome copies/rat diluted in 400μl PBS) were injected into a lateral tail vein.

Sun H., Hodgkinson C.P., Pratt R.E, & Dzau V.J. (2021). Crispr-Cas9 deletion of hepatic Angiotensinogen gene results in sustained control of Hypertension: A Potential for Cure?. Hypertension (Dallas, Tex. : 1979), 77(6), 1990-2000.

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Detailed Protocol for Constructing Expression Vectors

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References and construction details for all expression vectors are provided in Supplementary information, Table S2. Some expression vectors were constructed by in-fusion cloning using the Seamless Cloning Kit (Beyotime Biotechnology, Shanghai, China; Cat# D7010M). PCR-amplification reactions were performed using KOD One PCR Master Mix (Toyobo Inc., Osaka, Japan; Cat# KMM-201). Ligation reactions were performed using T4 DNA ligase (New England Biolabs, Beverly, MA, USA; Cat# M0202L). Restriction endonucleases were purchased from New England Biolabs. Unpublished plasmids pAdDeltaF6 (Addgene plasmid #112867) and pAAV2/8 (Addgene plasmid #112864) were gifts from James M. Wilson (University of Pennsylvania). pSG5-lamda4G was kindly provided by Matthias W. Hentze.^{55 (link)}

Shao J., Li S., Qiu X., Jiang J., Zhang L., Wang P., Si Y., Wu Y., He M., Xiong Q., Zhao L., Li Y., Fan Y., Viviani M., Fu Y., Wu C., Gao T., Zhu L., Fussenegger M., Wang H, & Xie M. (2024). Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells. Cell Research, 34(1), 31-46.

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