Frequently Asked Questions

question marks

Which viral vector should I use?

What is meant by "replication defective"?

What AAV Serotype should I use?

I had a custom virus made several years ago. How should I re-order this?

What buffer will the virus be in?

How are viral titers determined?

What quality control assays are performed on viral vectors?

What quality control is required for plasmids and why?

What are RCAs?

What is the Viral Vector Core's confidentiality policy?

Does the Viral Vector Core maintain stocks of virus?

Can I freeze and thaw the virus?

 

 

Which viral vector should I use?

Different virus lines are better for some applications than others. Choosing the best virus to meet your experimental goals can be difficult, but we are here to help. Below is a chart with a general overview of our viral vectors. If you have more detailed questions, please e-mail us at vectors@uiowa.edu or call us at (319) 335-6726 and we would be happy to discuss your experiment and how the Viral Vector Core can help your lab. 

Comparison of Vectors at a Glance

Vector

Infection

Advantages

Disadvantages

Expression

Expression Level

Insert Size

Titer

Adenovirus

(Serotype 5)
Dividing and non-dividing cells Non-integrating

Episomal expression

Wide host cell range

Immunogenic 
Transient High Up to 7.5 kb  1E+10 to 1E+11 pfu/ml
Helper Dependent Adenovirus*

(HDAd)     
Dividing and non-dividing cells      Non-integrating Episomal expression

Broad tissue tropism 

Immunogenic 

FGAd5 helper virus contamination 
Transient High Up to 27 kb 1E+10 to 1E+11 pfu/ml
Adeno-Associated Virus Dividing and non-dividing cells Episomal expression

Non-immunogenic

Non-pathogenic 
Stable Moderate ssAAV-up to 4.5kb

scAAV-up to 2.25Kb
1E+12 to 1E+13 vg/ml
Lentivirus*

(FIV and HIV) 
Dividing and non-dividing cells Random integration

Can be pseudotyped

Minimal immune response

FIV-Not a human pathogen 
Stable Moderate Up to 6.5kb 1E+7 to 5E+8 TU/ml
Retrovirus*

(MMuLV)
Dividing cells Integrates

Immunogenic 
Stable High Up to 6.5kb 1E+6 to 5E+7 TU/ml
Baculovirus** Insect cells Species-specific 1mg/1x109cells High No upper size limit 1E+7 to 5E+8 pfu/ml

*Replication defective.

** Replication defective in mammalian cells. Replication competent in insect cells.

 

What is meant by 'replication defective"?

Replication defective means that critical portions of the viral genome have been removed such that the viral vector can no longer replicate. The removal of these genes makes room for insertion of the genetic material that the investigator wants transduced into target cells. For production of viral vectors that can transduce target cells, the critical viral genes are expressed in trans in packaging cells. The particles that are generated from the packaging cells contain the recombinant replication defective genome.

The nature of the deletions that make the various vectors replication defective are explained under the description for each vector.

 

What AAV Serotype should I use?

A literature search is the best place to start. Pilot studies may be needed to test various serotypes in your experimental system. We offer a serotype testing kit with the CMVeGFP control package with serotypes: AAV2/1, AAV2/2, AAV2/4, AAV2/5, AAV2/6, AAV2/8 and AAV2/9. See the In-Stock Vector page. Below is a list of tropisms for various serotypes based on current literature.  

Serotype

Tropism

AAV1 Muscle, heart, CNS, eye, lung
AAV2 In vitro, CNS
AAV4 CNS, RPE
AAV5 Lung (airway, alveoli), eye, CNS
AAV6 Muscle, lung (airway), heart, adipose, liver
AAV8 Liver, muscle, eye, CNS, adipose
AAV9 Lung (alveoli), liver, muscle, heart CNS, adipose

 

I had a custom virus made several years ago. How should I re-order this?

Any previously produced virus can be ordered on the Reorder Custom Vectors by Aliquot order form in iLabs. You do not need to initiate a new construct project. If it has been many years since we last made the virus, we may ask you to provide the sequence and map of the construct to update our information in iLabs. We often need this information to maxi prep the plasmid and confirm the integrity by digest or to titer the virus by QPCR. We require a minimum purchase of at least 1ml for Adenovirus and 500ul for AAV or Lentiviral vectors to offset the cost of production.  Note: all prices are subject to change depending on customer type-contact us for a more accurate quote at Vectors@uiowa.edu.

  • Standard Titer Adenovirus: $1,051.00 per ml; titer range 1010 pfu/ml to 5 x 1010 pfu/ml.
  • High Titer Adenovirus: $2,871.00 per ml; titer range 8 x 1010 pfu/ml to 2 x 1011 pfu/ml.
  • Helper Dependent Adenovirus: $1,051.00 per ml; titer range 1010 pfu/ml to 5 x 1010 pfu/ml.
  • Adeno-associated virus: $2,376 per 500 µl; titer range 1012 vg/ml to 1 x 1013 vg/ml.
  • Lentivirus: $1,342.00 per 500 µl; titer range 107 TU/ml to 5 x 108 TU/ml.
  • Retrovirus: $1,342.00 per 500 µl; titer range 106 TU/ml to 5 x 107 TU/ml.
  • Baculovirus: please inquire...$370-$550 depending; sucrose concentrated stock titer range 109 pfu/ml to 1 x1010 pfu/ml.
  • Vaccinia: please inquire.

What buffer will the virus be in?

Different virus lines have different final buffers, please inquire for details.

Vector

Buffer

Adenovirus

(Serotype 5) 
A-195 or 3% Sucrose/PBS
Helper Dependent 

Adenovirus (HDAd)
A-195 
Adeno-Associated Virus Lutrol/F68/PBS
Lentivirus

(FIV and HIV)
4% Lactose/PBS
Retrovirus 4% Lactose/PBS
Baculovirus Sf9 media with 2% PBS

 

How are viral titers determined?

Titers are determined differently for each vector type and may use a combination of methods to assess the total number of particles (both live and dead/empty) or the total number of live “infectious” particles. See the details below for the standard titer units reported and the range expected for each vector.

Adenovirus titers are reported as an “infectious” or transduced titer in plaque forming units per ml (pfu/ml) determined by an immunostain of the Adenovirus hexon protein in transduced 293 cells. The concentration of viral DNA and protein is also measured in purified adenoviral preps at OD260 to determine the total number of recombinant adenovirus particles (pt/ml) and assess the ratio of “infectious” to “non-infectious” particles.

 Helper Dependent Adenovirustiters are given as an “infectious" or transduced titer in IGU/ml (Infectious Genomic Units/ml) determined by QPCR of genomic DNA extracted from transduced cells. HDAd vectors are also assessed for transduced titer by FACs when fluorescent reporters are available; this titer is available upon request.

AAV titers are given as a “physical” titer in viral genomes per ml (vg/ml) determined by direct QPCR of purified vector particles. Vectors are also assessed for transduced titer by FACs when fluorescent reporters are available. Due to serotype complications, not all transduced titers are comparable so they are therefore reported only when requested. All AAV titers are further confirmed by Silver Stain to check that the staining density agrees with the total particles loaded based on the vg/ml and to confirm the packaging envelope proteins are in the correct/functional ratios.

Lentiviral titers are given as an “infectious” or transduced titer in TU/ml (Transducing Units/ml) determined by QPCR of genomic DNA extracted from transduced HT1080 cells. Lentiviral vectors are also assessed for transduced titer by FACs when fluorescent reporters are available; this titer is available upon request.

Vector

Titer

Infectious Titer?

Units

Additional QC

Buffer

Adenovirus

(Serotype 5) 
1x1010to 5x1010pfu/ml Yes Plaque forming units per milliliter RCA assay A-195 or 3% Sucrose/PBS
Helper Dependent Adenovirus 1x1010to 5x1010pfu/ml Yes Infectious genomic units per mililiter RCA assay, QPCR for helper virus contamination levels, and FACS (when applicable) A-195
Adeno-Associated Virus 1x1012to 1x1013vg/ml No, physical titer Viral genomes per milliliter Silver Stain and FACs (when applicable) LutrolF68/PBS or 400mM NaCl/20mM Tris
Lentivirus

(FIV and HIV)
1x107to 5x108TU/ml Yes Transducing units per milliliter FACs (when applicable) 4% Lactose/PBS
Retrovirus

(MMuLV) 
1x106to 5x107pfu/ml Yes Transducing units per milliliter FACS (when applicable) 4% Lactose/PBS
Baculovirus 1x107to 5x108pfu/ml Yes Plaque forming units   Sf9 media with 2% FBS

 

What quality control assays are performed on viral vectors?

The vector core makes every effort to provide low endotoxin, sterile vectors suitable for research work in vitro and in vivo. Infectious titers are measured whenever possible to guarantee quality, functional virus is being produced. In addition, the Viral Vector Core checks internal catalog vectors for Luciferase and Cre Recombinase expression.

Ad5 Vectors: Quality control assays include a transduced titer in plaque forming units per ml, a physical measure of particles per mL, and an assay for replication competent adenovirus (RCA) contamination.

HDAd Vectors: Quality control assays include a transduced titer in plaque forming units per ml, a physical measure of particles per mL, an assay for replication competent adenovirus (RCA) and helper virus contamination, and FACs when appropriate.

AAV Vectors: Quality control assays include a physical titer (viral genomes/ml) by QPCR, transduced titer by FACs when appropriate, and silver stain to further assess virus titer and packaging efficiency.

Lentiviral Vectors: Quality control assays include a transduced titer by QPCR and transduced titer by FACs when appropriate.

 

What quality control is required for plasmids and why? 

We require gene information, a sequence, map, and quality control digest upon submission of all new projects. Every effort is made to deliver a high titer, high quality viral vector. We recommend that you sequence all plasmids. Despite best efforts, some genes of interest may confer cellular toxicity that results in lower vector titers. If problems are noted, we will work with investigators on a case by case basis to troubleshoot.

Ad5 and HDAd Plasmid Quality Control

One of the main rate limiting steps in Ad5 production is the quality of DNA for the initial transfection and recombination. We highly recommend that you use a good quality endotoxin free maxi prep. We can provide this optional service.

AAV Plasmid Quality Control

Because purifications in AAV may be greatly influenced by sequence, 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. AAV ITRs are bi-directional and may take the path of least resistance where the shuttle backbone is very small as compared to the transgene cassette leading to inappropriate packaging of the shuttle backbone. Reports have also been published of the inappropriate packaging of the Rep or Cap Sequences. J Virol. Jan 2003; 77(1): 776–781.Salvetti et al. Our testing indicates this is more common with small backbone AAV plasmids.
  • 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.

If problems do arise, investigators will be notified 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.

Lenti Plasmid Quality Control

One of the main rate limiting steps in Lentivirus production is the quality of DNA for the initial transfection and recombination. We highly recommend that you use a good quality endotoxin free maxi prep. We can provide this optional service.

 

What are RCAs?

Replication competent Adenoviruses (RCAs) are wild-type like viruses in a population of replication-deficient viruses. RCAs result from a crossover event where the deleted E1 region of the virus re-inserts itself into the virus while being grown in packaging cells. Once this happens, the RCA adenovirus will replicate without the need of a packaging cell line and outgrow recombinant adenovirus in packaging cells. To avoid the occurrence of RCA, viruses should not be serially propagated. Recombinant adenovirus with high RCA can be rescued by clonal isolation after plaque assay. 

 

What is the Viral Vector Core's confidentiality policy?

All investigator plasmids, sequences, and viruses are kept private and confidential. This is always our number one priority. However, investigators may or may not choose to allow us to give out their contact information for the purposes of inquiry and granting access to their vectors. Investigators also have the option of choosing to give broad or specific permission for certain vectors to other parties. In all instances, vectors will never be publically listed or made available to anyone else without specifically written, direct permission to do so which may likely involve a Material Transfer Agreement. 

 

Does the Viral Vector Core maintain stocks of viruses?

Some Adenovirus and HDAd catalog vectors are stored in 1 mL aliquots, but some vectors may be available in small aliquots (100uL).

AAV control stocks are available in 25 μL aliquots.

Lenti control stocks are available in 100 μL aliquots only.

For MMLV, Vaccinia and Baculovirus stocks, please inquire.

Investigator Viruses

All vector lines maintain an inventory aliquot of the original plasmid submitted from the investigator for vector construction and can reproduce the vector from this plasmid stock. In addition, Adenoviral and HDAd vectors maintain a glycerol stock of the viral vector that can be amplified more quickly than having to start from the plasmid recombination step. Similarly, Adeno-associated viral vectors produced by the Baculovirus system are maintained long term as a P1 or P2 baculovirus stock that can be readily amplified to AAV vector without repeating the transposition, transfection, and amplification steps.

 

Can I freeze and thaw the virus?

All of our vectors are titered after 1 freeze thaw cycle so that the titer will be equivalent to what you will use in your first experiment. We have tested the stability of vectors through multiple freeze and thaw cycles and find that the titer is consistent for approximately 3 freeze and thaw cycles before it begins to drop; however, we recommend that investigators limit freeze/thaw cycles to as few as possible. Vectors are fairly stable for short periods of 24-48 hours at 4 degrees. Storage instructions can be found on the Technical Resources page. We recommend that the vector be stored at -80 upon receipt. Thaw the vector only when you are ready to use it and make aliquots of no less than 25 μl in 0.5 ml tubes.