The shuttle vector for making recombinant adenoviruses containing the GADD45G gene (Ad-GADD45G) was synthesized based on Gateway technology using ViraPower Adenoviral Gateway Expression Kit (Invitrogen) as per the manufacturer’s instructions. The human GADD45G full-length open reading frame (ORF) clone (Ultimate ORF clone, Invitrogen) was provided in an entry vector (Gateway pENTR221, Invitrogen). The GADD45G ORF was cloned into the destination vector pAd/CMV/V5-DEST by using LR Clonase II enzyme (Invitrogen) to synthesize pAd/CMV/V5-GADD45G. pAd/CMV/V5-GADD45G was then digested with Pac I enzyme and transfected into HEK293A cells using Lipofectamine 2000 (Invitrogen) to produce recombinant adenoviruses (Ad-GADD45G). For use as controls, pAd/CMV/V5/lacZ for β-galactosidase expression was provided with the kit and recombinant adenoviruses containing the lacZ gene (Ad-LacZ) were generated. The shuttle vector for making recombinant adenoviruses expressing hemagglutinin (HA)-tagged MAGEH1 proteins (Ad-HA-MAGEH1) was synthesized by Applied Biological Materials (Ferndale, WA) For purification and concentration, the Adeno-X maxi purification kit (Takara, Mountain View, CA) was used. For titration, HEK293A cells infected with recombinant adenoviruses were detected using an antibody specific for the adenovirus hexon protein with the Adeno-X rapid titer kit (Takara).
Adeno x maxi purification kit
Manufactured by Takara Bio
Sourced in United States
The Adeno-X Maxi Purification Kit is a laboratory equipment product designed for the purification of recombinant adenovirus particles. The kit provides a streamlined process for concentrating and purifying adenoviral vectors from cell culture supernatants or lysates.
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Lab products found in correlation
Adeno x rapid titer kit, by Takara Bio (14 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Pad cmv v5 dest, by Thermo Fisher Scientific (3 mentions)
Virapower adenovirus expression system, by Thermo Fisher Scientific (2 mentions)
Gateway system, by Thermo Fisher Scientific (2 mentions)
Pspax2, by Cellecta (2 mentions)
Pmd2 g, by Cellecta (2 mentions)
Polyethylenimine (pei), by Merck Group (2 mentions)
Paxcawtit, by Takara Bio (2 mentions)
Hek293, by RIKEN BioResource Center (2 mentions)
Virapower adenoviral expression system, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Lr clonase 2 enzyme, by Thermo Fisher Scientific (1 mentions)
Virapower adenoviral gateway expression kit, by Thermo Fisher Scientific (1 mentions)
Ultimate orf clone, by Thermo Fisher Scientific (1 mentions)
Pad pl dest, by Thermo Fisher Scientific (1 mentions)
Lr clonaseii, by Thermo Fisher Scientific (1 mentions)
Pentr directional topo cloning kit, by Thermo Fisher Scientific (1 mentions)
31 protocols using adeno x maxi purification kit
1
Adenoviral Vector Construction for GADD45G
2
Generating Recombinant Adenoviruses for EGFP Expression
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Replication-incompetent adenoviruses (type 5) were generated using the Virapower adenovirus expression system (Invitrogen) following previous reports.14 (link) Site-specific DNA recombination between the entry vector (pENTR-EUI-EGFP or pSIQ-EGFP) and the adenoviral destination vector pAd/PL-DEST (Invitrogen) was performed using LR clonase Ⅱ (Invitrogen) to generate pAd-EUI-EGFP or pAd-SIQ-EGFP. The pAd-EUI-EGFP or pAd-SIQ-EGFP plasmids were linearized by Pac 1 (New England Biolabs) digestion and then transfected into 293A cells using Lipofectamine 2000 (Life Technologies) to produce recombinant adenoviruses. Cells were grown until 80% cytopathic effect was observed, and then they were harvested to prepare recombinant adenovirus. The adenovirus particles were subsequently amplified in 293A cells, yielding crude lysates containing adenovirus particles, which were purified using the Adeno-X Maxi Purification Kit (#631533, Takara Bio). HaCaT cells were infected with Ad/EUI-EGFP or Ad/SIQ-EGFP in combination with the administration of Teb. Subsequently, the cells were analyzed by fluorescence microscopy, and cell lysates were harvested for western blot analysis.
3
Adenovirus Generation Using ViraPower System
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Adenoviruses were generated using the ViraPower Adenovirus Expression System (Invitrogen). The transgene between ITRs of the AAV vector was subcloned into the pENTR/D-TOPO vector using a pENTR Directional TOPO Cloning Kit (Invitrogen). The DNA inserts were transferred to the pAd/CMV/V5-DEST vector via LR reaction with a Gateway System (Invitrogen). The plasmids were digested with Pac I (New England BioLabs), purified and transfected into subconfluent 293A cells using Lipofectamine 3,000 (Invitrogen). Then 293A cells were cultured for 10–12 days with replacement of the medium every 2 days. The cells and culture medium were harvested, freeze-thawed twice, and centrifuged to obtain the adenovirus-enriched supernatants. To amplify adenoviruses, the aliquots of the supernatants were added to cultured 293A. After 2 – 4 amplifications, the adenovirus-containing media were purified using an Adeno-X Maxi Purification Kit (TaKaRa). Viral titers were determined by a plaque-forming assay using 293A cells according to the manufacturer’s instruction.
4
Adenovirus-Mediated Multi-Promoter Amplification
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Adenovirus expressing PCA3-3STA, PSEBC-TSTA and SV40-Luc were generated as previously described 23 (link). To obtain the multi-promoter integrated two-step transcriptional amplification system, PCA3-Cre-PSEBC-ITSTA, in a single backbone containing the Cre recombinase and the TSTA system with the stop cassette, the above constructed pENTR-L1R5 and pENTR-L5R2 backbone plasmids were subcloned into pAd-pL-DEST by LR cloning with LR clonase II Plus enzyme (Invitrogen, Carlsbad, CA, USA). Adenoviral backbones containing plasmids were transfected into 293A cells for the viral production. Amplified virus particles were column purified using Adeno-X™ Maxi purification kit (Takara) and stored in buffer A195 after buffer exchange 29 (link). Titration for each of the viruses was done using Adeno-X™ Rapid Titer Kit (Takara).
5
Adenovirus-Mediated Expression of APE1/Ref-1 Variants
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Adenoviruses encoding‐galactosidase (Adβ‐gal), full‐length human APE1/Ref‐1 (Ad‐APE1/Ref‐1), or PPTLS‐APE1/Ref‐1 (Ad‐PPTLS‐APE1/Ref‐1) with secretory signal sequences were prepared according to previously described methods,
16 (link),
17 (link),
18 (link) and their treatment effects were compared. Human embryonic kidney‐293 T cells (HEK293T) with the large T‐antigen of Simian virus 40 (ATCC, Manassas, VA, USA) were used to amplify and purify the adenoviruses. HEK293T cells were seeded in plates at 2.5 × 105 cells/mL and maintained at 37°C in a humidified incubator with 5% CO2. HEK293T cells were infected with 500 multiplicity of infection (particle‐forming units per cell) of the adenovirus containing Adβ‐gal, Ad‐APE1/Ref‐1, or Ad‐PPTLS‐APE1/Ref‐1 and incubated for 48 h. The adenovirus was purified using the Adeno‐X maxi Purification Kit (Takara Bio, San Jose, CA, USA). The cell culture‐amplified adenovirus was quantified using an Adeno‐X rapid titre kit (Takara Bio). APE1/Ref‐1 sandwich enzyme‐linked immunosorbent assay (ELISA; MediRedox, Daejeon, Korea) was used to quantify secretory APE1/Ref‐1 in the culture supernatant or whole‐cell lysates, as previously described.
19 (link)
16 (link),
17 (link),
18 (link) and their treatment effects were compared. Human embryonic kidney‐293 T cells (HEK293T) with the large T‐antigen of Simian virus 40 (ATCC, Manassas, VA, USA) were used to amplify and purify the adenoviruses. HEK293T cells were seeded in plates at 2.5 × 105 cells/mL and maintained at 37°C in a humidified incubator with 5% CO2. HEK293T cells were infected with 500 multiplicity of infection (particle‐forming units per cell) of the adenovirus containing Adβ‐gal, Ad‐APE1/Ref‐1, or Ad‐PPTLS‐APE1/Ref‐1 and incubated for 48 h. The adenovirus was purified using the Adeno‐X maxi Purification Kit (Takara Bio, San Jose, CA, USA). The cell culture‐amplified adenovirus was quantified using an Adeno‐X rapid titre kit (Takara Bio). APE1/Ref‐1 sandwich enzyme‐linked immunosorbent assay (ELISA; MediRedox, Daejeon, Korea) was used to quantify secretory APE1/Ref‐1 in the culture supernatant or whole‐cell lysates, as previously described.
19 (link)
6
Adenoviral and Lentiviral Plasmid Construction
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Adenoviral plasmids for PSEBC-TSTA and CMV-TSTA were previously described [31 (link)]. Lentivirus-expressing Renilla luciferase and GFP used in LNCaP cells were constructed using the plasmid pccl-CMV-RL-IRES-EGFP [33 (link)]. Adenoviral backbone plasmids were transfected into 293A cells for viral production. Amplified virus particles were column purified using Adeno-X™ Maxi purification kit (Takara Bio USA, Mountain View, CA, USA) and stored in buffer A195 after buffer exchange [40 (link)]. Titration of each virus was performed using Adeno-X™ Rapid Titer Kit (Takara).
7
Adenoviral Transduction of Cardiomyocytes
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A human-codon optimized bPAC gene was synthesized (GeneArt, ThermoFisher, Waltham, MA) from the available bPAC sequence (accession number: GU461306.2) and followed with a cMyc-derived epitope, an internal ribosomal entry site (IRES) sequence and mCherry. The sequence of bPAC-cMyc-IRES-mCherry was carried in pShuttle-CMV vector for generation of adenovirus (AdbPAC) in 293AD cells with the AdEasy system (Agilent Technologies, Santa Clara, CA) (34 (link)). The adenovirus carrying the GFP gene under the CMV promoter (AdGFP) was obtained from the Baylor College of Medicine Vector Development Core (Houston, TX). Purification and titer determination of the adenoviral particles were carried out using the Adeno-X™ Maxi Purification Kit (Takara Bio USA, San Jose, CA) and QuickTiter™ Adenovirus Titer ELISA Kit (Cell Biolabs, San Diego, CA), respectively. The adenovirus propagation was conducted by transducing 293AD cells at 50% confluence with harvested when the cytopathic effect is complete. The cardiomyocytes were transduced at MOI as stated at 24 hours after seeding. Following an incubation period of 48 h with viral particles, fresh medium was replenished, and assays were carried out 24 h after.
8
PDMS-Mediated Fibronectin Patterning and Virus Transduction
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Polydimethylsiloxane (PDMS) stamps were created to microcontact print fibronectin in patterns of tangential 1600 µm 2 circles on 40 kPa gels. was harvested 48 hours after transfection, and the viral particles were precipitated using Lenti-X concentrator (Takara Bio) following the manufacturer's protocol. Low passage HLMVECs were transduced with concentrated virus, along with 10µg/mL Polybrene (Millipore Sigma).
Expression was observed 2-7 days following infection.
Adenovirus was used to express VE-Cadherin-GFP in HLMVECs (Shaw et al, 2001). The virus was amplified in HEK293AD cells, by transducing the cells and collecting the virus containing supernatant, once the cytopathic effect was complete. The viral particles were then purified using the Adeno-X Maxi Purification Kit (Takara Bio) as per the manufacturer's instructions. Low passage HLMVECs were transduced with the virus, and expression was observed 2-3 days following infection.
Expression was observed 2-7 days following infection.
Adenovirus was used to express VE-Cadherin-GFP in HLMVECs (Shaw et al, 2001). The virus was amplified in HEK293AD cells, by transducing the cells and collecting the virus containing supernatant, once the cytopathic effect was complete. The viral particles were then purified using the Adeno-X Maxi Purification Kit (Takara Bio) as per the manufacturer's instructions. Low passage HLMVECs were transduced with the virus, and expression was observed 2-3 days following infection.
9
Lentiviral and Adenoviral Transduction Protocols
10
Adenoviral Constructs for Nuclear Receptor Studies
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