Custom Adenovirus Constructs

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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: $2,734 for 2ml standard titer. New Custom Vectors from amplified particles: $2,332 for 2ml standard titer. Re-order Custom Vectors from amplified particles: $1,051 for 1ml standard titer. Contact vectors@uiowa.edu for high titer or small aliquot pricing.

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

$2,332 per vector plus shipping and handling. Additional 1ml aliquots can be purchased at $1051 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 except for shipping after a Material Transfer Agreement is signed. 

Sample Required

60μg 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. A diagnostic digest of the plasmid will also be required: a single digest each with BssHII, SacII, XmaI and one that linearizes, either PacI or NheI. You can choose to have us do the digest for you for $50 per 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 or AdenopureTM filter purification method.
  • 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.

Vehicle

Adenovirus vectors are re-suspended in A 195 buffer (see reference below).

Cost

CsCl banding: $2,734 per construct (plus shipping and handling)
Additional 1ml aliquots can be purchased at $1051 per ml. These may take 4-6 weeks for a new prep to be made.

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 diagnostic digest 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 provide the plasmid prep service to you for $290. We recommend that you sequence all plasmids and that you functionally test 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.