New Custom Vectors from recombination: $3,852 for the yield from 40 plates with a minimum of 2 x 1ml standard titer. Listed price is for non-profit institutions.
Adenoviral vectors are a versatile research tool with a number of positive aspects. The recombinant viruses can infect dividing and non-dividing cells, support transient, high-level protein expression, and are also easily amplified and purified to high concentrations (1x1010pfu/mL-1x1011pfu/mL). They can accommodate inserts of up to 8 kb under normal circumstances. For larger inserts, we recommend HDAd.
Photo by Russell Kightley
The University of Iowa VVC developed and currently uses the RAPAdTM system (U.S. Patent No. 6,830,920 B2). A description of the system can be found in "A simple method for the rapid generation of recombinant adenovirus vectors" published in Gene Therapy (https://www.nature.com/articles/3301197). This system eliminates many of the challenges of standard adenoviral production. The RAPAdTM construction plasmids have left-hand ITR, E1a, and partial E1b sequence deletions which greatly reduces wild type occurrence in the final vector preparations. There is, therefore, no need for time-consuming plaque purification steps. All virus preps are tested for replication competent adenovirus (RCA) contamination by immuno-staining procedures.
The RAPAdTM system uses a two plasmid transfection system for initial viral rescue, the viral shuttle plasmid and the viral backbone plasmid. The viral shuttle plasmid contains the gene of interest that the recombinant virus will produce, as well as a few viral protein sequences. The viral backbone contains the remaining viral proteins as well as a region of homology to the viral shuttle plasmid. After linearization and transfection into HEK293 cells (which provide the needed E1 and E3 proteins), the plasmids undergo homologous recombination to produce a full length adenoviral genome with the gene of interest inserted into the E1 region. The VVC has a number of backbones with additional E3 deletions that have reporter genes (i.e. eGFP, Beta-gal, mCherry, etc) inserted into them or that can be used for inserts larger than 8kb. Using viral backbones with reporters already inserted into the E3 region allows for reporter gene function without any additional cloning. This system also offers the possibility of expressing two separate proteins with custom made backbones. For more information contact vectors@uiowa.edu.
The University of Iowa VVC can produce adenovirus from other plasmid systems.
Learn More About Adenovirus Products
Our Adenovirus is available in regular and high titer.
All of our controls are available in 1mL aliquots, but only some are available in 100uL aliquots.
This virus will be provided in A-195 buffer.
| Titer | infectious units / mL | Volume |
| Regular | 1e+10 to 7e+10 pfu/ml | 1,000uL |
| 100uL* | ||
| High | 8e+10 to 3e+11 pfu/ml | 1,000uL |
| 100uL |
Interested in Ordering?
In-Stock Orders
Browse/order our Adenovirus In-Stock Products
*Listed prices are for non-profit customers only, additional fees may apply.
Custom Vector Orders
***Custom vector orders need to be submitted in iLabs.
Vectors are stocked based on supply and demand and may not be readily available but can be made "Upon Request". Most vectors with a catalog number are kept in stock but please call 319-335-6726 or email vectors@uiowa.edu for the latest availability as stock amounts are always changing.
Please allow 4-10 weeks for delivery of out-of-stock or "Upon Request" vectors.
We are also able to offer a number of vectors utilizing commonly studied genes of interest with permission or a three-way MTA from the original investigator. Please inquire.
Interested in Ordering?
Please view our ordering information for custom constructs to learn how to register for an account, place orders for in-stock vectors and make payment through our online system.
Pricing
New Custom Vectors from recombination: $3,852 for the yield from 40 plates with a minimum of 2 x 1ml standard titer. New Custom Vectors from amplified particles: $3,303 for the yield from 40 plates with a minimum of 2 x 1ml standard titer. Re-order Custom Vectors from amplified particles: $1,350 for 1ml standard titer. Contact vectors@uiowa.edu for high titer or small aliquot pricing. Pricing listed is for external academic non-profit customers. Please contact us for a specific quote.
Description
Viral vector construction is performed using the RapAdTM System developed by the University of Iowa VVC (For description, refer to from the article "A simple method for the rapid generation of recombinant adenovirus vectors" published in Gene Therapy 7:1034-1038, 2000). Adenovirus vectors prepared in the core are E1 and E3 deleted. They have a total E1a deletion (*m.u 1.4 to 4.5) plus a partial E1b deletion (*m.u. 4.7 to 9.2). These deletions are what make the vector replication deficient. They also have a partial E3 deletion, 720bp for the sub360 backbone, a 1.6 kb deletion for the dl309 backbone and a 3.1 kb deletion for the total E3 deleted backbone.
*m.u = Map units
Characteristics
- Episomal gene expression.
- Infects dividing and non-dividing cells.
- Transient high-level protein expression.
- Accommodates inserts of up to 7.5kb. Larger inserts can be added, provided that an equivalent part of the viral genome has been properly deleted.
- High viral titer can be produced: 1 x 1010 pfu/ml to 1 x 1011 pfu/ml.
Disadvantages and Adverse Effects
Elicits host immune response, thus depleting the number of transduced cells over time in-vivo.
Short-term expression of the transgene due to lack of integration into the host genome.
Reamplifying Existing Adenoviral Vector
Amplification, purification and titer from viral particles
Sample Required
Viral vector particles approximately 50-100μl of purified virus or 500μl-1ml of viral lysate is sufficient.
Service Details
- Large-scale amplification.
- Purification by double cesium chloride gradient.
- With every new lot an infectious titer (pfu/ml) is provided.
- Each lot is checked for replication competent Adenovirus (RCA) contamination.
Material Provided
2 ml of ready to use virus in 1mL aliquots at a concentration of at least 1x1010 pfu/ml.
Vehicle
Adenovirus vectors are provided in biological buffer A195 buffer (see reference below).
Cost
$3,303 per vector plus shipping and handling. Additional 1ml aliquots can be purchased at $1,250 per ml.
Producing New Adenoviral Vector from a Shuttle Plasmid
Recombination, amplification, purification and titer
Shuttle
A cloning/expression adenovirus shuttle vector is provided free of charge when intended for production at the UI VVC.
Sample Required
200μg of Adenovirus plasmid expressing the gene of interest at a concentration greater than 0.25μg/μl. We also require a sequence, map, and specific gene of interest information in accordance with our biosafety protocols. Full plasmid sequencing is required. Please confirm that either PacI or NheI is unique and linearizes the shuttle plasmid.
Timeline
6-10 weeks from the time the plasmid is received.
Service Details
- Transfection and recombination
- Large-scale amplification
- Purification by double cesium chloride gradient.
- With every new lot an infectious titer (pfu/ml) is provided.
- Each lot is checked for wild-type virus contamination (RCA).
Material Provided
2 ml of virus in 1mL aliquots at a concentration of at least 1x1010 pfu/ml. Small aliquots are available upon request for an additional fee.
Vehicle
Adenovirus vectors are re-suspended in A 195 buffer (see reference below).
Cost
CsCl banding: $3,852 per construct (plus shipping and handling)
Shuttle Vectors
Please see the shuttle plasmids page for a complete list of available shuttle vectors.
References
RapAdTM System: Anderson RD, Haskell RE, Xia H, Roessler BJ, Davidson BL. “A simple method for the rapid generation of recombinant adenovirus vectors”. Gene Ther. 2000 Jun;7(12):1034-8.
A195 Buffer: Evans RK, Nawrocki DK, Isopi LA, Williams DM, Casimiro DR, Chin S, Chen M, Zhu DM, Shiver JW, Volkin DB. Development of stable liquid formulations for adenovirus-based vaccines. J Pharm Sci. 2004 Oct;93(10):2458-75.
Plasmid Quality Control upon Submission
Every effort is made to deliver a high titer, high quality viral vector. We require gene information, a sequence, map, and a full plasmid sequencing upon submission of a new project. High quality supercoiled, endotoxin-free, shuttle plasmid DNA is critical to the success of the initial transfection and recombination. We are able to outsource the plasmid prep service to Genscript by quote. We recommend functionally testing the plasmid before submission.
Despite best efforts, some genes of interest may confer cellular toxicity that results in lower vector titers. We will notify investigators of progress and discuss the next steps if problems are noted.
The University of Iowa VVC provides shuttle plasmids at no cost when intended for vector production. Please email Vectors@uiowa.edu for sequence files.
Plasmid Backbone
The pacAd5 backbone was developed in the laboratory of Dr. Beverly L. Davidson and is patented as the RAPAdTM system (U.S. Patent No. 6,830,920 B2). A description of the system can be found in "A simple method for the rapid generation of recombinant adenovirus vectors," published in Gene Therapy 7:1034-1038, 2000. This system eliminates many of the problems of standard adenoviral production. The RAPAdTMconstruction plasmids have left-hand ITR, E1a, and partial E1b sequence deletions, which greatly reduces wild type occurrence in the final vector preparations.
Cloning Adenovirus Plasmids
The RapAdTM System uses a two plasmid transfection system to generate recombinant adenovirus particles, the shuttle plasmid and the viral backbone plasmid:
- The Shuttle Plasmid: The shuttle plasmid is usually cloned by the investigator and contains the gene of interest driven by the desired promoter and any other elements needed for a single or bicistronic construct. Reporter genes such as eGFP may be cloned into this plasmid or may come from the viral backbone plasmid. See the advantages and disadvantages below. The VVC offers shuttle plasmids with a number of common promoters already prepared and are available free of charge. The VVC also offers limited cloning services for investigators.
- The Viral Backbone Plasmid: This plasmid contains most of the adenoviral genome, including the partially (or fully) deleted E3 region. With a portion of the E3 deleted, an additional gene cassette can be added. The VVC offers several very convenient viral backbone plasmids with and without reporters for investigators to choose from. No cloning of these plasmids is necessary unless you have a custom design in mind. Simply choose the desired backbone when ordering the virus construction.
Insert Size
Adenovirus vectors can accommodates inserts of up to 7.5kb, this includes promoters, gene(s) of interest, and other elements. Larger inserts can be accommodated though, provided that an equivalent part of the viral genome has been properly deleted. The VVC currently has two backbones with additional portions of the E3 region deleted (known as the G0685 pacAd5 (9.2-100)∆1.6KbE3 and G0686 pacAd5 (9.2-100)∆3.1KbE3).
E1 Shuttle Plasmids
G0687 pacAd5mcspA
- Cat: G0687
- pacAd5 backbone, multiple cloning site, SV40 polyadenylation signal
G0688 pacAd5CMVmcspA
- Cat: G0688
- pacAd5 backbone, CMV (Cytomegalovirus) Promoter, multiple cloning site, SV40 polyadenylation signal
G0689 pacAd5RSVmcspA
- Cat: G0689
- pacAd5 backbone, RSV (Rous sarcoma virus) Promoter, multiple cloning site, SV40 polyadenylation signal
G0407 pacAd5CAGmcspA
- Cat: G0407
- pacAd5 backbone, CAG Promoter (CMV enhancer fused to the chicken beta-actin promoter), multiple cloning site, SV40 polyadenylation signal
G0796 pacAd5TREtightmcspA
- Cat: G0796
- pacAd5 backbone, TRE tight (TET Response Element) Promoter, multiple cloning site, SV40 polyadenylation signal
G0864 pacAd5hSyn1mscpA
- Cat: G0864
- pacAd5 backbone, tissue-specific human synapsin I promoter, multiple cloning site, SV40 polyadenylation signal
G0951 pacAd5mcswtIRESeGFPpA
- Cat: G0951
- pacAd5 backbone, multiple cloning site, wild-type IRES (internal ribosome entry site), eGFP (enhanced green fluorescent protein), SV40 polyadenylation signal. The IRES-eGFP functions as a non-fusion protein reporter without the addition of a second promoter.
G0950 pacAd5CMVmcswtIRESeGFPpA
- Cat: G0950
- pacAd5 backbone, multiple cloning site, wild-type IRES (internal ribosome entry site), eGFP (enhanced green fluorescent protein), SV40 polyadenylation signal. The IRES-eGFP functions as a non-fusion protein reporter without the addition of a second promoter.
G1059 pacAd5mcswtIRESmCherrypA
- Cat: G1059
- pacAd5 backbone, multiple cloning site, wild-type IRES (internal ribosome entry site), mCherry, SV40 polyadenylation signal. The IRES-mCherry functions as a non-fusion protein reporter without the addition of a second promoter.
G1071 pacAd5CMVmcswtIRESmCherrypA
- Cat: G1071
- pacAd5 backbone, CMV (Cytomegalovirus) Promoter, multiple cloning site, wild-type IRES (internal ribosome entry site), mCherry, SV40 polyadenylation signal. The IRES-mCherry functions as a non-fusion protein reporter without the addition of a second promoter.
G1072 pacAd5CAGmcswtIRESmCherrypA
- Cat: G1072
- pacAd5 backbone, CAG (CMV enhancer fused to the chicken beta-actin Promoter) Promoter, multiple cloning site, wild-type IRES (internal ribosome entry site), mCherry, SV40 polyadenylation signal. The IRES-mCherry functions as a non-fusion protein reporter without the addition of a second promoter.
G0996 pacAd5mU6miSafeSV40pA
- Cat: G0996
- pacAd5 backbone, Mouse U6 Promoter driving a scrambled miRNA control miSafe, Poly T termination signal, and SV40 polyadenylation signal.
- Please reference: Boudreau, R. L., et al. (2011). "Rational Design of Therapeutic siRNAs: Minimizing Off-targeting Potential to Improve the Safety of RNAi Therapy for Huntington's Disease." Mol Ther 19(12): 2169-2177.
G0997 pacAd5mU6shSafeSV40pA
- Cat: G0997 (scrambled control for use with G0455)
- Ad5 backbone, Mouse U6 Promoter driving a scrambled shRNA control shSafe, Poly T termination signal, and SV40 polyadenylation signal.
- Please reference: Boudreau, R. L., et al. (2011). "Rational Design of Therapeutic siRNAs: Minimizing Off-targeting Potential to Improve the Safety of RNAi Therapy for Huntington's Disease." Mol Ther 19(12): 2169-2177.
G1272 pacAd5CMVCas9SV40pA
- Cat: G1272
- Ad5 backbone, CMV Promoter driving human spCas9, and SV40 polyadenylation signal. MluI or AgeI estriction sites for gRNA cassette addition.