Viral Vector Production Unit FAQs

Which viral gene delivery system is the best to use for my experiments, adenovirus or AAV?

 

– What AAV serotype should I use? 

 

What adenovirus serotype should I use?

 

– Is it possible to express transgenes in specific cells?

The use of cell type specific promoters allows driving the expression of therapeutic genes in particular cell types only. For example: SYN and CamKII promoters are specific for neurons.

 

What buffer will the virus be in?

The delivery buffer depends on the recombinant virus ordered. But, you can specify a particular buffer on request. Click the link below for more information (link table). (es refereix a la taula d’abaix, de la següent pregunta)

 

How are viral titers determined?

The UPV determine viral titers according to 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 table below.

Vector Typical Titer Infectious Titer? Units QC Assays Additional QC Buffer
Adenovirus 1.5×1012 PP/mL  or 1×1011 IU/mL Yes Physical Particles or Infectious Units per milliliter Enzyme-linked immunoassay

Mycoplasma test

Sterility test

RCA assay PBSCaMg +10% glycerol
Adeno-Associated Virus 7×1012vg/ml No Viral genomes per milliliter Fluorimetric quantification (Picogreen)

Mycoplasma test

Sterility test

RCA assay Iodixanol + 40% PBSMK

– What quality control assays are performed on viral vectors?

The UPV uses methods to guarantee vector productions are sterile. Our vectors are suitable for research work in vitro and in vivo. Infectious titers are measured whenever possible to ensure that quality, functional virus is being provided to clients.

 

  • Ad Vectors:Quality control assays include an Enzyme-linked immunoassay, a Mycoplasma and Sterility tests.
  • AAV Vectors:Quality control assays include a Fluorimetric quantification (Picogreen), a Mycoplasma and Sterility tests.

 

– What is the turnaround time for virus production?

The typical turnaround time for virus production depends on vector type:

  • Ad Vectors: 6 weeks (without cloning).
  • AAV Vectors: 4-6 weeks (without cloning).

 

– What is the final volume of the virus? / How much virus do I get?

The final volume of virus delivered depends on vector type and the final concentration:

Ad Vectors: 2-3mL

AAV Vectors: 0.5-1.5 mL

 

– What is the required biosafety level for using recombinant viruses?

The AAV vectors produced in the UPV are classified as Biosafety Level (BSL) I and Adenovirus and Canine vectors are classified as BSL II. However, the nature of the transgene insert may alter their BSL classification. Detailed Safety Data can be found here. (link a Material Safety Data Sheet que estan a la pestanya Quality and Biosafety)

 

– What are RCAs?

Replication Competent Adenoviruses (RCAs) are wild-type like viruses that have the E1 region and can replicate without the need of a packaging cell line. An extra control assay for RCA is available. This test does not apply for AAV vectors.

 

– What are the recommended storage conditions for recombinant viruses?

We recommend that you place  s at -80°C on arrival and for long term storage.

 

– Can I freeze and thaw the virus?

We recommend that researchers limit freeze/thaw cycles to as few as possible. We have tested the stability of vectors through multiple freeze and thaw cycles and find that the titer is consistent for approximately 3 cycles before beginning to drop. For best results, thaw the virus on ice and prepare storage aliquots of the amount you typically use per experiment.

 

– What is the cost of virus production?

The cost of production depends primarily on factors such as the virus type (AAV or Ad), the scale of production (number of cells that must be grown, transfected or infected to produce the virus), the number of cloning steps required to generate the viral construct, extra quality tests required, etc.  For standard scale production orders the prices may vary between 1300 and 2100 euros per batch.

 

– How do I order a viral vector?

To order a viral vector please complete a Request Form (a separate form for each viral vector ordered) and send it back to us either by e-mail or fax. Please, see the viral vector request guidelines on page 2 in Request Form.1

 

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

Any previously produced virus can be re-ordered on the Request Form. You just need to indicate the name (and production number) of the previously produced viral vector and the scale of production desired.

 

– What quality control is required for plasmids and why?

We require gene information, a sequence or a map, upon submission of all new projects, in order to perform quality controls of the starting material.

Despite making every effort to produce a high titer, high quality viral vector, some genes of interest may have cytotoxic effects, resulting in lower vector titers. If problems are noted, we will contact the customer to explain how this may affect titer and timings.

Plasmid Quality Control

One of the main rate limiting steps in viral vector production is the quality of DNA for the initial transfection and recombination. We highly recommend that you use a good quality maxi prep, and in addition for AAV vectors, you should check the presence of both ITRs by restriction enzyme digestion before submission of an order. This is especially important regarding plasmids you may have obtained from another lab.

AAV Quality Control

Before approving and starting a new AAV production, we need to carefully review your order in the following situations:

  1. Constructs that are larger than the packaging capacity. Capacity is ~4.7 kb from ITR to ITR for single stranded AAV and ~2.2 kb from ITR to ITR for self-complementary AAV.
  2. Use of a novel or modified capsid.

 

-What types of viruses are offered by the core?

The two main viral vectors provided by the UPV are adeno-associated virus (AAV), and adenovirus (Ad). These two vectors are suitable for a wide range of genetic manipulations in a variety of tissues, in vitro and in vivo. More information is available in the Section “Types of Viruses”. Please send questions about these viruses to the core e-mail address (u.pvectors@uab.es).

 

– What is the optimal vector dose I should use for in vitro and in vivo experiments?

The optimal dose needs to be determined empirically for every experiment. For pilot studies, the vector including marker genes such as GFP, ßgal or luciferase is available at the UPV.

For in vitro studies adenovirus doses usually range between 10pp – 5,000pp per cell, and AAV doses between 500vc – 100,000vc per cell. For in vivo studies doses by intravenous administration range between 1x10E12 – 5x10E13 pp/kg for both, adenovirus and AAV vectors. [pp stands for physical particles; vc stands for viral copies]