Aav helper free system

Manufactured by Agilent Technologies

Sourced in United States

The AAV Helper-Free System is a set of plasmids designed for the production of recombinant adeno-associated virus (rAAV) particles without the use of a helper virus. The system includes the necessary components for packaging the desired rAAV gene of interest into viral particles.

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Lab products found in correlation

Sybr green, by Takara Bio (4 mentions) Elisa, by Progen Biotechnik (4 mentions) Hek293 cell, by American Type Culture Collection (4 mentions) Amicon ultra 30 k filters, by Merck Group (3 mentions) Paav mcs, by Agilent Technologies (2 mentions) Paav hrgfp, by Agilent Technologies (2 mentions) Amicon ultra 30k, by Merck Group (2 mentions) Geneart, by Thermo Fisher Scientific (2 mentions) Benzonase nuclease, by Merck Group (2 mentions) Amicon filter, by Merck Group (2 mentions) Viratrap aav purification kit, by Biomiga (1 mentions) Psilencer 2.1 u6 neo, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions) Control shrna lentivirus sc 108080, by Santa Cruz Biotechnology (1 mentions) Hek293h cells, by Thermo Fisher Scientific (1 mentions) Aavanced concentration reagent, by System Biosciences (1 mentions) Hct116, by Thermo Fisher Scientific (1 mentions) Dld 1, by Thermo Fisher Scientific (1 mentions) Ht1080, by Thermo Fisher Scientific (1 mentions) Facsdiva software version 5, by BD (1 mentions)

43 protocols using aav helper free system

Recombinant AAV2/5 for Astrocyte-Specific Expression

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Recombinant AAV2/5 was prepared by using AAV Helper-Free System (Agilent Technology) with some modification. To generate an AAV2/5 capable of expressing mCherry-PKI fusion protein in astrocytes, AAV2/5 rep, and cap plasmid was purchased from Penn Vector Core (University of Pennsylvania) and pZac2.1 gfaABC1D-tdTomato^{61 (link)} was a gift from Baljit Khakh (Addgene plasmid # 44332). To construct pZac2.1 gfaABC1D-mCherry–PKI, the cDNA for mCherry-PKI was amplified from p4mt-mCherry-PKI that was kindly provided by Dr. Giulietta Di Benedetto^{62 (link)} by using following primers; Forward 5’-AAGCTTGAATTCGCCACCATGGTGAGCAAGGGCGAGGAGGA-3’ and reverse 5’-AAGCTTGCGGCCGCCTATGACTCGGACTTAGCAG-3’. The tdTomato portion of pZac2.1 gfaABC1D-tdTomato was replaced with mCherry-PKI at EcoR I and Not I site. After DNA sequence validation, these plasmids were used for AAV2/5 production. Viruses were prepared according to manufacturer’s instruction (AAV Helper-Free System; Agilent Technology) and purified by a column chromatography using a ViraTrap AAV purification kit (Biomiga) as described in the manufacturer’s protocol. Viral titers were determined using a quantitative real-time PCR method. The both AAV2/5 titers were 2.0 × 10¹² GC/ml and transduction was performed to ONH astrocytes with 400,000 MOI.

Shim M.S., Kim K.Y., Bu J.H., Nam H.S., Jeong S.W., Park T.L., Ellisman M.H., Weinreb R.N, & Ju W.K. (2018). Elevated intracellular cAMP exacerbates vulnerability to oxidative stress in optic nerve head astrocytes. Cell Death & Disease, 9(3), 285.

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Gene Targeting in HCT116 Cells

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Gene targeting vectors were constructed by using the recombinant adeno-associated virus (rAAV) system (24 (link)). Briefly, two homologous arms flanking exon 2 of Bim or Noxa, along with a neomycin-resistant gene cassette (Neo), were inserted between two Not I sites in the AAV shuttle vector pAAV-MCS (Agilent Technologies). Packaging of rAAV was performed by using the AAV Helper-Free System (Agilent) according to the manufacturer’s instructions. HCT116 cells containing two copies of Bim and Noxa were infected with the rAAV and selected by G418 (0.5 mg/ml, Mediatech) for 3 weeks. Drug-resistant clones were pooled and screened by PCR for targeting events with the following primers for Bim: P1, 5’-TGATTGGATGTATTCAGAGG/P2, 5’-CATTACTACAGCACTCTCCTC, or for Noxa: P1, 5’-GCGAAGGAAGTGGTGCATTG/P2, 5’-CTGAAGAAAACAGATGTGAGG, in combination with a primer for Neo 5’-TCTTGACGAGTTCTTCTGAG/5’-TTGTGCCCAGTCATAGCCG. Prior to targeting the second allele, the Neo cassette, which is flanked by Lox P sites, was excised from a heterozygous clone by infection with an adenovirus expressing Cre recombinase (Ad-Cre). Single clones were screened by PCR for Neo excision using P1/P2, and two independent positive clones were infected again with the Bim- or Noxa-targeting construct. After the second round, Neo was excised by Ad-Cre infection, and gene targeting was verified by PCR and western blotting.

Tong J., Zheng X., Tan X., Fletcher R., Nikolovska-Coleska Z., Yu J, & Zhang L. (2018). Mcl-1 phosphorylation without degradation mediates sensitivity to HDAC inhibitors by liberating BH3-only proteins. Cancer research, 78(16), 4704-4715.

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Generating Plasmids for Marmoset D1R and D2R Knockdown

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AAV vectors were based on the AAV helper-free system (Agilent Technologies, CA, USA). To generate plasmids encoding shRNA driven by a U6 promoter, a 0.7-kb PvuII fragment from pSilencer 2.1-U6 NEO (Ambion Life Technologies, MA, USA) was inserted into the blunted MluI site of pAAV-hrGFP (Agilent Technologies)52 (link). The CMV promoter for hrGFP was replaced by the human synapsin I promoter. A shRNA cassette was inserted into the plasmid digested with BamHI and HindIII located directly below the U6 promoter52 (link). The shRNA target sequence for the marmoset D1R was 5′-GTCGAATGTTCTCAACCAGAA-3′, and that for the marmoset D2R was 5′-GGACAGACCTCACTACAATTA-3′. Silencing efficacy was evaluated using the Dual-Luciferase reporter assay system (Promega, WI, USA)53 (link). The shRNAs with more than 80% knockdown efficacy in vitro were identified (see Supplementary Fig. 1), and those having high efficacy were tested more than twice.

Takaji M., Takemoto A., Yokoyama C., Watakabe A., Mizukami H., Ozawa K., Onoe H., Nakamura K, & Yamamori T. (2016). Distinct roles for primate caudate dopamine D1 and D2 receptors in visual discrimination learning revealed using shRNA knockdown. Scientific Reports, 6, 35809.

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Efficient Viral Transduction of GSK-3β Variants

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Lentiviruses used for knocking down GSK-3β and its control were obtained from Santa-Cruz (GSK-3β shRNA lentivirus: sc-270460-V; control shRNA lentivirus: sc-108080), and Adeno-associated viruses used for knocking in of phosphorylation-resistant form (S9A) of GSK-3β were generously provided by Dr. Yoon et al.^{85 (link)}. The viruses were produced by using AAV Helper-Free System (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's recommended protocol. Briefly, pAAV expression vector, pAAV-RC (DJ) and pHelper were transfected into AAV-293 cells. They were further incubated for 66–72 h. Then cells were harvested and went through repeated freeze/thaw cycles. The debris were centrifuged and the supernatant was mixed with 40% (vol/vol) PEG (to final 10% PEG) and left at 4 °C for 36–48 h. The viral particles were obtained from further centrifugation and resuspension in ice-cold PBS for two times. The GSK-3β S9A [MSGRPRTTAFAES…] containing plasmid was constructed from an original vector pAAV-ef1a-DIO-eYFP (from Karl Deisseroth, addgene #27,056, Water Town, MA, USA). The final form is pAAV-ef1a-S9A-P2A-eYFP where P2A is a linker peptide that is cleaved post-translationally, and the cleavage produces functional S9A and eYFP proteins. The viral titer (~ 1.0 × 10⁸ infectious unit/µl) was measured in HT-1080 cells via flow cytometry.

Song S., Kim J., Park K., Lee J., Park S., Lee S., Kim J., Hong I., Song B, & Choi S. (2020). GSK-3β activation is required for ZIP-induced disruption of learned fear. Scientific Reports, 10, 18227.

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Cloning of Precursor Sequence in AAV Vector

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The precursor sequence was retrieved from the NCBI database (Gene ID: 723886). Restriction sites of KpnI and XhoI enzymes were placed upstream and downstream of the gene, respectively. The gene was synthesized by Biomatik Company (Ontario, Canada), received in the BSK vector, and subcloned in the pAAV-2-MSC-eGFP vector (a human AAV-2 vector included in the AAV Helper Free System designed by Agilent Technologies, USA, which had been previously constructed in our laboratory adapted from Ref. [41 ]). Sequence of this vector provided in Supplementary Table S1. Ligation and transformation in Escherichia coli XL-10 were performed as described previously [42 (link)].

Zolfaghari N., Soheili Z.S., Samiei S., Latifi-Navid H., Hafezi-Moghadam A., Ahmadieh H, & Rezaei-Kanavi M. (2023). microRNA-96 targets the INS/AKT/GLUT4 signaling axis: Association with and effect on diabetic retinopathy. Heliyon, 9(5), e15539.

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AAV Helper-Free Virus Purification

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According to a previously published protocol (28 (link)), we packaged the AAV helper-free system (Agilent Technologies, Santa Clara, CA), the pRep-Cap (AAV5; Applied Viromics, Fremont, CA), and the pHelper plasmid. AAV was purified using a cesium chloride density gradient, combined with ultracentrifugation. Virus titer was determined using quantitative real-time polymerase chain reaction analysis (SYBR green; Takara Bio Inc., Shiga, Japan).

Obi-Nagata K., Suzuki N., Miyake R., MacDonald M.L., Fish K.N., Ozawa K., Nagahama K., Okimura T., Tanaka S., Kano M., Fukazawa Y., Sweet R.A, & Hayashi-Takagi A. (2023). Distorted neurocomputation by a small number of extra-large spines in psychiatric disorders. Science Advances, 9(23), eade5973.

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Recombinant AAV Vector Production

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The recombinant AAV vectors were produced by calcium phosphate transfection of human embryonic kidney cells (HEK 293-H cells, Thermo Fisher Scientific Waltham, MA, USA) using a three-plasmid based production protocol (AAV helper free system; Agilent Technologies, Santa Clara, CA, USA) and purified as described [99 (link)] Vector yield was analyzed by ddPCR, and viral titers were determined by ELISA (Progen, Heidelberg, Germany) according to the manufacturer’s manual.

Kuklik J., Michelfelder S., Schiele F., Kreuz S., Lamla T., Müller P, & Park J.E. (2021). Development of a Bispecific Antibody-Based Platform for Retargeting of Capsid Modified AAV Vectors. International Journal of Molecular Sciences, 22(15), 8355.

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Production of AAV8 Viral Vectors

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Adeno associated virus of the serotype 8 (AAV8-hSyn-sDarken) were produced in house. HEK-T-cells were transfected with the help of the AAV Helper-Free System (Agilent) and after three days harvested and concentrated the Virus with the AAVanced™ Concentration Reagent (System Biosciences).

Kubitschke M., Müller M., Wallhorn L., Pulin M., Mittag M., Pollok S., Ziebarth T., Bremshey S., Gerdey J., Claussen K.C., Renken K., Groß J., Gneiße P., Meyer N., Wiegert J.S., Reiner A., Fuhrmann M, & Masseck O.A. (2022). Next generation genetically encoded fluorescent sensors for serotonin. Nature Communications, 13, 7525.

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AAV Vector Production and Purification

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The AAV Helper-Free System (Agilent Technologies, Inc., Santa Clara, CA, USA) was used to produce AAV and purify the AAV vectors^{43 (link)}. The plasmids pAAV-hSyn-FLEX-hM3Dq-mCherry, pAAV-hSyn-FLEX-mCherry and pAAV-EF1α-DIO-hChR2(E123T/T159C)-EYFP were purchased from Addgene (ID: 44361, 44362, 50459, 35509). pAAV-CMV-FLEX-hrGFP was constructed starting with the pAAV-hrGFP plasmid (Agilent Technologies), and pAAV-CMV-FLEX-DTA was constructed starting with the pAAV-MCS plasmid (Agilent Technologies), and its cell-specific ablation was confirmed as previously reported^{44 (link)}. The injection volume was 600 nl per side.

Kato Y., Katsumata H., Inutsuka A., Yamanaka A., Onaka T., Minami S, & Orikasa C. (2021). Involvement of MCH-oxytocin neural relay within the hypothalamus in murine nursing behavior. Scientific Reports, 11, 3348.

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Gene Editing with SaCas9 and AAV9

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pZac2.1-ESYN-SaCas9 was constructed from the cis-cloning plasmid pZac2.1 with ESYN promoter and pENTER-SaCas9 (Tabebordbar et al., 2016 (link); Koga et al., 2020 (link)). Synthetic oligonucleotides included the targeting sequence for Cacna2d1 Exon 10 (5′-ATCTCGGAGACAGATGTTCGG-3′) or oligonucleotides without target sequence (control) were substituted with the targeting site in the original pENTER-U6-sgBsa1 plasmid (Ran et al., 2015 (link)). The resulting U6-sgRNA cassettes (U6-sgCacna2d1 or U6-sgControl) were transferred into pZac2.1-ESYN-mCherry plasmid. AAV9-ESYN-SaCas9, AAV9-ESYN-mCherry-U6-sgCacna2d1 and AAV9-ESYN-mCherry-U6-sgControl were produced using the AAV Helper-Free System (Agilent Technologies) as reported previously (Sano et al., 2020 (link)). AAV9-EF1α-FLEX-mCherry (Kohro et al., 2020 (link)) was kindly gifted from Prof. Tsuda laboratory. The used viral titers were as follows: AAV9-ESYN-SaCas9, 2.0 × 10¹² genome copies (GC)/ml; AAV9-ESYN-mCherry-U6-sgCacna2d1 and AAV9-ESYN-mCherry-U6-sgControl, 0.5 × 10¹² GC/ml; AAV9-EF1α-FLEX-mCherry 1.0 × 10¹² GC/ml.

Koga K., Kobayashi K., Tsuda M., Kubota K., Kitano Y, & Furue H. (2023). Voltage-gated calcium channel subunit α2δ-1 in spinal dorsal horn neurons contributes to aberrant excitatory synaptic transmission and mechanical hypersensitivity after peripheral nerve injury. Frontiers in Molecular Neuroscience, 16, 1099925.

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