Storage and Handling
- General recommendations
Do not thaw and re-freeze the purified vectors more than once. If the tube contains 25 µl, make 5 to 10 µl aliquots and re-freezing immediately at -80°C. To do this, thaw the vector on ice for a few minutes, then aliquot into pre-chilled tubes (on dry ice). CAV-2 vectors are in PBS without Ca++ or Mg++ and 10% glycerol. The vector are stable for years at -80°C. If you want to dilute and aliquot the vector, use the same buffer (PBS w/o Ca++ or Mg++ and 10% glycerol).
N.B. If you see a precipitate in the tube (because of the drop in pH at –80°C) it is likely the vector is no longer ideal for in vivo gene transfer studies but can be used for in vitro assay. Once an aliquot is diluted in media (eg DMEM) containing serum, it can be stored at –20°C and then thawed and re-frozen at least 4 or 5 times without significant loss of infectivity.
- Can we centrifuge stock tubes ?
You can centrifuge CAV-2 vectors at 3000g, 5 min, 4°C to avoid losing a few microleter.
- Can we keep purified CAV-2 vectors at 4°C, and how long ?
This is not recommended, we don’t know the half-life of CAV-2 vectors in these conditions.
- In case of in vivo injections, can we re-use CAV-2 vectors that stayed on ice?
We recommend to use these “thawed” vectors for in vitro work after they sat on ice. Mix them with medium and FCS and store at -20°C.
- CAV-2 vector biosafety
Wild type CAV-2 does not propagate in primate cells (Klonjkowski HGT 1997).
Wild type CAV-2 and CAV-2 vectors were tested to see if it could co-propagate with human adenoviruses (type 5) and the results were negative. However, one can never exclude the possibility.
- Are replication-competent CAV-2 particles contaminating your stocks ?
Replication-competent CAV-2 vectors have never been detected in CAV-2 vector preps (see Kremer et al JVI for assay sensitivity 1 pp/1x10e11 pp sensitivity).
The E1-transcomplementing cell line and vectors do not contain a region of overlap, which theoretically precludes replication-competent virus generation by homologous recombination (Kremer et al JVI 2000).
E1-deleted CAV-2 vectors have no known danger to any animal species.
Wild type CAV-2 is used as a vaccine for all dogs in western countries. It is highly likely that all of us have all been exposed to CAV-2. However, CAV-2 neutralizing antibodies are extremely rare in humans which suggest that CAV-2 does not propagate in humans (Perreau Mol Ther).
CAV-2 vectors are deleted in the E1 region, which codes for transactivating factors.
Without the E1 region, CAV-2 vectors cannot propagate in any (including canine) cells, but can be internalized by cells that express its receptor (CAR) Soudais et al JVI 2001.
To produce E1-deleted CAV-2 vectors, a cell line constitutively expressing the E1 region was created (Kremer et al JVI 2001 & Soudais et al Mol Ther 2001). These cells (DKZeo cells) can propagate E1-deleted CAV-2 vectors. For a comparison, DKZeo cells are the canine equivalent of human PER.C6 cells (Fallaux HGT 1998).
CAV-2 vectors expressing non-oncogenic proteins are classified as P1/A1 in the French system.
- Will these CAV-2 vectors be dangerous for my dog ?
No, they can’t propagate in your dog – or cat, skunk, bear, ferret, bat, fish, kangaroo, wolf, panda, monkey, chimp, brother, sister, son, daughter or cousin.
And, unfortunately, not even in your mother-in-law.
About CAV-2 constructs
- What promoter do CAV Cre and CAV GFP have ?
CMV early promoter (+ SV40 polyA).
- Does CAV Cre express a fluorophore ?
We have a CAV Cre-GFP vector available.
- Which of your papers do you prefer us to cite for the fact that CAV-2 infects neurons from axon terminals ?
Soudais et al FASEB 2001. Here we showed sensory, motor and central neuron transduction with CAV vectors in vivo.
- Is there a strong preference for CAV-2 to infect axon terminals vs. axons-in-passage? Do they infect from axon-in-passage at all?
Based on all our results, and observations by others, the most efficient uptake is likely at axon terminals in vivo. Some uptake by axons-in-passage may occur.
What we currently believe is that the coxsackievirus adenovirus receptor (CAR) is the principle means of uptake and it is preferentially located at presynaptic termini. Other pathways might exist, but in CAR KO mice, CAV-2 vector transduce very few cells.
- How long does it take to see expression ?
Less than 24 hours.
- How long will transgene expression last ?
Depends on the animal model (mice < rats < monkeys) and strain. Cre doesn’t need long-term expression though. GFP can be from 2 weeks to >6 months.
- How much should we inject?
We recommend injecting no more than 1 x 10e9 pp per coordinate during stereotactic delivery in rodents. You will probably have to dilute the vector you will receive.
- Example of calculation : I received an aliquot of 25 µL with a titer of 10 x 10e12 pp/mL (= 10 x 10e9 pp/µL) and my volume of injection is 500 nL. I have to dilute the aliquot 5 times (working titer = 2 x 10e9 pp/µL). I add 100 µL of PBS +10% glycerol to the original tube and aliquot in smaller volume before refreezing.
- Am I correct that you only deliver CAV-2 vectors in a titre of 2.5 x 10e12 pp/ml and the available quantities are 100 µL (2.5 x 10e11 pp), 500 µL (1.25 x 10e12 pp) or 1mL (2.5 x 10e12 pp) ?
The 2.5 x 10e12 pp/mL on the vector request form is an example. As the titer of each prep varies, you have to select a quantity (pp) of virus and not a volume or a concentration. For example, our latest prep of CAV Cre has a titer of 4.6 x 10e12 pp/mL, according to your request you will receive x tubes of 25 µL of CAV Cre.
- We need to add you to our institute supplier list, can we send you the form to fill in and return to us ?
No. We are not a company. The vector core is housed in a normal research lab at a French institute that is overwhelmed with requests. Therefore, we created a way to help you to get our vectors. We don’t have the time, the personnel, or the legal right to fill in and sign these forms for every (usually American) university. Many ingenious colleagues and administrative staff have found ways around this form. If you wait for us to fill this in, you’ll never get our vectors.
- How long does it take to process the order and ship it out ?
The bottleneck is normally how fast you get the MTA and signed PO returned to us. We normally ship the Monday after we receive all the paperwork.
- Do you take USD ?
Yes, if you do the correct conversion from euros ont the PO.
- Can you accept credit cards ?
No, the CNRS has not found a way to do this. Payment can be done either by purchase order (and wire transfert upon reception of invoice) or if your institution can’t edit PO by prepayment via wire transfer.
- Do you take international order ?
Yes, we’ve sent vectors to every continents (except Antartica).
- How much is the charge for shipping ?
We use your DHL, FedEx or WorldCourier express mail account and we send by priority.
If you don’t have or can’t give us an express mail account, we’ll use our DHL account.
N.B.: We can’t ship via DHL to Canada and Asia.
For Australia, the most reliable courier is WorldCourier.
Most importantly, we are not responsible for the package once it leaves our institute.