Triple Transfection System- $2,600 for non-profit institutions.

AAV is a single-stranded DNA parvovirus that commonly infects humans but has no known pathology. Recombinant adeno-associated virus has a wide host range, long-term expression in vivo, and low immunogenicity. Recombinant AAV is a promising viral vector for many gene transfer applications.

Tropism determined by choice of capsid serotype.

Long-term expression in terminally differentiated cells.

Wide range of cells can be infected, including non-dividing cells.

The size of the transgene cassette is limited to ~4.7 kb for non-self complementary vectors and ~2.2 kb for self-complementary vectors. The optimal size of an AAV package is approximately 3.5kb. Larger packages can be produced but may have lower titers. 

Photo by Russell Kightley

The University of Iowa VVC offers research grade, high quality AAV vectors with titers from 5 x1011 to 1 x1013 vg/ml. AAV vectors are prepared by a triple plasmid transfection in HEK-293 cells. The cis-acting AAV plasmid carries the AAV expression cassette containing the gene of interest flanked by the AAV inverted terminal repeats (ITR). The genes for the functional and structural proteins, rep and cap, respectively, and the adenovirus helper genes are provided in trans. The dual or triple baculovirus system for AAV is available for specific research projects. Please contact vectors@uiowa.edu.

Purified vector preparations are subjected to a variety of quality control measures including a physical ddPCR titer to asses viral genomes, transduced titer by FACS when reporters are available, and silver stain to further assess titer and viral purity. Control stocks are assayed for Cre recombinase and Luciferase activity.

The VVC currently offers the following serotypes: 1, 2, 5, 6, 8, 9, DJ, 6.2, PHP.B, PHP.eB, PHP.S and rh10. Our nomenclature refers to the structural Rep proteins from AAV2 followed by the capsid serotype, for example AAV2/5. Novel peptide modified serotypes can also be produced on a case-by-case basis. Purification methods are customized for each serotype and may require optimization for novel capsids. Additional charges may apply for novel capsids.

Pilot studies may be needed to determine which serotype will work best in your system. We offer a serotype kit that includes 25 µl of AAV2/1, 2/2, 2/5, 2/6, 2/8, 2/9, 2/DJ, 6.2, 2/PHP.B, 2/PHP.eB, 2/PHP.S and 2/rh10 under the In-Stock Vector Page.

For more information on AAVDJ, please see:  Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA, Kay MA. In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol. 2008;82:5887–5911.

For more information on AAVrh10, please see: Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A. 200; 99(18):11854-9.

Serotype Tropism
AAV1 Muscle, Heart, CNS, Eye, Lung
AAV2 In vitro, CNS
AAV3 Human Liver Cancer Cells
AAV4 CNS, RPE
AAV5 Lung (airway, alveoli), Eye, CNS
AAV6 Muscle, Lung, Heart, Adipose, Liver
AAV8 Liver, Muscle, Eye, CNS, Adipose
AAV9 Lung (alveoli), Liver, Muscle, Heart, CNS, Adipose
 AAVDJ In vitro, Liver, Heart, Kidney
AAVrh10 Pleura, CNS
AAVPHP.B CNS, Heart, Muscle
AAVPHP.eB CNS, Muscle
AAVPHP.S CNS, Heart, Muscle

Learn More About AAV Products

Our naming designation refers to the structural Rep proteins from AAV2 followed by the capsid serotype, for example AAV2/5. 

AAV vectors are offered in the following serotypes: AAV2/1, AAV2/2,  AAV2/5, AAV2/6, AAV2/6.2, AAV2/8, AAV2/9, AAV2/DJ, AAV2/rh10, PHP.B, PHP.eB, PHP.S.  

Vectors are provided in F68/PBS.  F68/PBS storage buffer does not require dialysis and is biocompatible with 0.001% Lutrol/F68 (poloxamer 188) to prevent aggregation of virus.

Titer (viral genomes / mL) Volume
~5e+11 to 5e+13 vg/mL 25uL

 

Order In-Stock AAV Vectors

Triple Transfection System- $2,600

The vectors are prepared by a triple plasmid transfection in HEK-293 cells. The cis-acting AAV plasmid carries the AAV expression cassette containing the gene of interest flanked by the AAV2 inverted terminal repeats (ITRs). A second plasmid provides the genes for the structural proteins, rep and cap. The third pHelper plasmid provides the adenovirus helper genes. The triple transfection system works with any AAV plasmid from any source. 

Material provided: 500 µl in 100 µl aliquots (Titer: 5x1011 to 1x1013 vg/ml (viral genomes/ml). Additional aliquots may be ordered with a minimum order of 500ul.

Serotypes: AAV2/1, AAV2/2, AAV2/5, AAV2/6, AAV2/6.2, AAV2/8, AAV2/9, AAV2/DJ, AAV2/rh10, PHP.B, PHP.eB, and PHP.S. Modified capsids are considered on a case by case basis. 

Shuttle: A cloning/expression AAV shuttle vector is provided after a Material Transfer Agreement is signed. Other outside pAAV shuttle vectors are also compatible with this system.

Sample required: 1000 µg of high-quality, transfection-grade AAV plasmid expressing the gene of interest at a concentration greater than 0.50 µg/µl with an A260/280 ratio of at least 1.80.

Sample Plasmid Quality Control: We require full plasmid sequencing. Every time an AAV shuttle plasmid is amplified, recombination can occur with the Inverted Terminal Repeats (ITRs) of the plasmid preventing the cassette from being packaged into virus. There are several companies that do this rapidly in a cost-effective manner. Some of these companies have a dropbox available on many US campuses.

Suggested additional Plasmid Quality Control:  Prior to sending sample for sequencing, we suggest digests to look for integrity within the ITRs. Please load a sample of at least 300ng of the DNA per well to get the best results. Suggested digests as follows: 

  1. Lane 1 : Clearly Labeled Molecular Weight Ladder
  2. Lane 2 : Uncut 
  3. Lane 3 :  BssHII single digest 
  4. Lane 4 : MscI single digest
  5. Lane 5 : XmaI or SmaI single digest

Timeline: 3-4 weeks from the time the plasmid is received depending on the current queue and serotype.

Services include:

  • Transfection.
  • Large-scale amplification of AAV vectors.
  • Purification performed using a variety of methods depending on the serotype. Most often this is an iodixanol gradient, followed by either an ion-exchange filter membrane or affinity chromatography. CsCl purification is also commonly used.
  • Quality control assays include a physical titer (vg/ml) by ddPCR, transduced titer by FACS when appropriate, and silver stain to further assess virus titer and purity.
  • Vehicle: The vectors are provided in F68/PBS

Sample Quality Screening and Disclaimer

Every effort is made to deliver a high titer, high quality viral vector. Viral vector preps are repeated a second time if they do not pass our quality control at no charge, provided that the plasmid submitted meets several criteria. We require gene information, a sequence, map, and ITR digest upon submission of a new project.

Preps will not be repeated and charges will apply to the first prep regardless of outcome in the following situations:

  • Constructs that are over packaging capacity. This is ~4.7 kb from ITR to ITR for single stranded AAV and ~2.2 kb from ITR to ITR for self-complementary AAV.
  • Where the shuttle backbone size is equal to or smaller than the ITR to ITR transgene. 
  • Involve a novel or modified capsid.
  • Constructs where the sequence does not match the ITR digest. We highly suggest that you perform your own ITR digest before submission of a project. This is especially important on plasmids you may obtain from another lab. 

Investigators will be notified when constructs are flagged as problematic and we will discuss the pros, cons, and finances of proceeding with the project. Sometimes the gamble to proceed with plasmids that fall under these criteria are warranted and we will do our best to guide you in the decision process.

Despite best efforts, some genes of interest may confer cellular toxicity that results in lower vector titers. We will notify investigators of progress and problems and discuss the next step if problems are noted.

Ordering Custom Adeno-Associated Virus Constructs

Shuttle plasmids are provided free of charge when intended for virus production at the University of Iowa Viral Vector Core. Please contact Vectors@uiowa.edu for sequence files.

 

Non-Self Complementary Shuttle Plasmids

ITR to ITR cassette size is limited to ~4.7kb

G0202 pFBAAVmcsBgHpA

pFBAAV backbone, multiple cloning site, Bovine Growth Hormone polyadenylation signal


G0347 pFBAAVCMVmcspBgHpA

pFBAAV backbone, CMV (cytomegalovirus) promoter, multiple cloning site, bovine growth hormone polyadenylation signal


G0261 pFBAAVRSVmcsBgHpA

pFBAAV backbone, RSV (rocus sarcoma virus) promoter, multiple cloning site, bovine growth hormone polyadenylation signal


G0345 pFBAAVCAGmcsBgHpA

pFBAAV backbone, CAG promoter (CMV enhancer fused to the chicken beta-actin promoter), multiple cloning site, bovine growth hormone polyadenylation signal


G0746 pFBAAVmcswtIRESeGFPBgHpA

pFBAAV backbone, multiple cloning site, wild type IRES (internal ribosome entry site), eGFP (enhanced green flourescent protein), bovine growth hormone polyadenylation signal. The IRES-eGFP functions as a non-fusion protein reporter without the addition of a second promoter.


G0692 pFBAAVCMVmcswtIRESeGFPBgHpA

pFBAAV backbone, CMV (cytomegalovirus) promoter, multiple cloning site, wild-type IRES (internal ribosome entry site), eGFP (enhanced green fluorescent protein), bovine growth hormone polyadenylation signal. The IRES-eGFP functions as a non-fusion protein reporter without the addition of a second promoter.


G0747 pFBAAVCAGmcswtIRESeGFPBgHpa

pFBAAV backbone, CAG promoter (CMV enhancer fused to the chicken beta-actin promoter), multiple cloning site, wild-type IRES (internal ribosome entry site), eGFP (enhanced green fluorescent protein), bovine growth hormone polyadenylation signal. The IRES-eGFP functions as a non-fusion protein reporter without the addition of a second promoter.


G1066 pFBAAVmcswtIRESmCherryBgHpA

pFBAAV backbone, multiple cloning site, wild-type IRES (internal ribosome entry site), mCherry (enhanced green fluorescent protein), bovine growth hormone polyadenylation signal. The IRES-mCherry functions as a non-fusion protein reporter without the addition of a second promoter.


G1068 pFBAAVCMVmcswtIRESmCherryBgHpA

pFBAAV backbone, CMV (cytomegalovirus) promoter, multiple cloning site, wild-type IRES (internal ribosome entry site), mCherry, bovine growth hormone polyadenylation signal. The IRES-mCherry functions as a non-fusion protein reporter without the addition of a second promoter.


G1067 pFBAAVCAGmcswtIRESmCherryBgHpA

pFBAAV backbone, CAG promoter (CMV enhancer fused to the chicken beta-actin promoter), multiple cloning site, wild-type IRES (internal ribosome entry site), mCherry, bovine growth hormone polyadenylation signal. The IRES-mCherry functions as a non-fusion protein reporter without the addition of a second promoter.


G0358 pFBAAVmcsCMVhrGFPSV40pA

pFBAAV backbone, multiple cloning site, CMV (cytomegalovirus) promoter driving hrGFP (Humanized Renilla reniformis green fluorescent protein), SV40 polyadenylation signal.


G0619 pFBAAVmcsCMVeGFPSV40A

Extended mcs. pFBAAV backbone, multiple cloning site (more available sites), CMV (cytomegalovirus) promoter driving eGFP (enhanced green fluorescent protein), SV40 polyadenylation signal


G0760 pFBAAVmU6mcsCMVeGFPSV40pA

pFBAAV backbone, mouse U6 promoter (RNA polymerase III), multiple cloning site designed for RNAi insertion, poly T termination sequence, CMV (cytomegalovirus) promoter driving eGFP (enhanced green fluorescent protein), SV40 polyadenylation signal


G0774 pFBAAVmU6mcsCMVmCherrySV40pA

pFBAAV backbone, mouse U6 promoter (RNA polymerase III), multiple cloning site designed for RNAi insertion, poly T termination sequence, CMV (cytomegalovirus) promoter driving mCherry (red fluorescent protein), SV40 polyadenylation signal


G1006 pFBAAVmU6miSafeCMVmCherrySV40pA

Scrambled control for testing use with cat: G0774.

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.


G0784 pFBAAVmU6miSafeCMVeGFPSV40pA

Scrambled control for testing use with cat: G0760.

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.


G1060 pFBAAVTREmcsBgHpACMVrtTAwtIRESeGFPSV40pA

pFBAAV backbone, TRE (TET response element) promoter, multiple cloning site designed for RNAi insertion, bovine growth hormone polyadenylation signal, CMV (cytomegalovirus) promoter driving tetracycline-controlled transactivator (rtTA-M2), wild-type IRES (internal ribosome entry site), eGFP (enhanced green fluorescent protein), SV40 polyadenylation signal. This plasmid was designed for inducible expression (TET-ON) of the miRNA cassette in the presence of the antibiotic tetracycline (doxycycline).


G1061 pFBAAVTREmiSafeBgHpACMVrtTAwtIRESeGFPSV40pA

Scrambled control for testing use with cat: G1060.

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.


G0013 pFBGR

pFBAAV backbone, CMV (cytomegalovirus) promoter and P10 insect promoter driving eGFP (enhanced green fluorescent protein) , SV40pA polyadenylation signal.

 

 

Self-Complementary Shuttle Plasmids

ITR to ITR cassette size is limited to ~2.2kb.

G0463 pscAAVmcsBgHpa

Self-complementary plasmid for use with the triple transfection system only. Multiple cloning site, bovine growth hormone polyadenylation signal. The backbone is based on self-complementary AAV vectors kindly provided by Brian Kasper.


G0605 pscAAVmCMVmcsBgHpA

Self-complementary plasmid for use with the triple transfection system only. Modified CMV (cytomegalovirus) promoter, multiple cloning site, bovine growth hormone polyadenylation signal. The backbone is based on self-complementary AAV vectors kindly provided by Brian Kasper.

Ordering Adeno-Associated Virus Shuttle Plasmids