rAVE Frequently Asked Questions. 

Please read below for answers to some of our most "frequently asked questions". If your question still isn't answered after browsing our FAQ please click here. 

How are rAVE products shipped? Why is shipping so expensive? 
Why should I incorporate an haemaglutinin (HA) tag sequence?
What dose of rAVE should I be using for my experiments?
How stable are rAVE vectors? How should they be stored?
What's the difference between physical and genomic particles?
What is the difference between genomic titer and infectious titer?
Do you have an MSDS for the rAVE products?
Is there a limit to the size of the gene that can be delivered by rAVE?
My gene of interest is slightly larger than the 1600bp limit for incorporating into your standard rAVE vector? Is there an alternative vector that can be used?
Why do you recommend the use of mannitol in delivery of rAVE to the brain?

Question not covered by FAQ?

How are rAVE products shipped? Why is shipping so expensive? 

Innovative global delivery methods save you money.

Although future advances in lyophilization techniques may one day allow for shipment of our rAVE products at room temperature, currently these products require shipment at temperatures below 4ºC. Until recently the only feasible way to send these products to you would be to ship using a large amount of dry ice of approx 10kg weight (to the US as an example or 30kg to Europe). There are significant hurdles with this method including cost. To ship a product globally with dry ice using a specialized shipping company will cost approximately USD$1000 making this a prohibitively expensive method. Dry ice itself is a hazardous material. Alternatively while we could appoint in-country distributors to handle the distribution of our products this would also significantly add to the end cost of our products.

New advances in shipping technology however allow for the direct global fulfilment of perishables by GeneDetect without the use of dry ice and allow us to bring down the total cost of shipping your rAVE products to you to USD$499 which includes the cost of customs fees and logistics expenses! This cost is "per shipment" meaning it pays to consolidate multiple rAVE orders into one shipment to save on costs.

GeneDetect has extensively tested the Deep Chill™ Shipper System. Using our packaging methods we are able to maintain rAVE samples at a temperature of 0-1ºC over 7 days!! 



Actual test results using an internal temperature probe.

Why should I incorporate an haemaglutinin (HA) tag sequence?

A frequent problem researchers have is, how am I going to be able to distinguish gene expression produced by my vector from endogenous gene expression especially if endogenous gene expression is high or worse there are no commercial antibodies available to my protein. How do I know the transgene is being expressed? We’ve solved this problem for you by including options to incorporate a haemaglutinin (HA) tag sequence either to the N-terminal (i.e. 5´) or the C-terminal (i.e. 3´) end of your gene of interest. The HA tag enables easy detection and the ability to distinguish endogenous from transgene expression. HA is a commonly used protein surveillance tag. At 10 amino acids in length it has little or no effect on the normal physiological function of the tagged protein, and the presence of this tag on the transgene product of your rAVE vector means through the use of an HA-specific antibody tracking of the protein is simple and efficient. This is extremely useful when using rAVE vectors on tissue high in endogenous transgene expression. To detect the HA tag we recommend the HA.11 mouse monoclonal antibody produced by BAbCO.

This depends on the application. Generally speaking, the degree of transgene expression will correlate directly to the amount of vector administered. Factors such as 'incubation' time, tissue or cell type, and transgene all effect expression levels. For transducing cultured cells in vitro (100-200µl of media per well) we typically find that 1-2µl of a vector stock (at 5 x 10¹⁰ genomic particles per ml) added to the media (1:100 dilution) will infect and express in 30-75% of the treated cells within 3 days, depending on cell type and transgene being used.

For in vivo applications, again the application will dictate the dose, and should be determined by the investigator. As a guide, for infusion into the brain we typically inject 2µl. To improve spread of the vector at the injection site co-infusion with mannitol is recommended. For more detailed information regarding in vivo transduction refer to our protocols or the following reference: Mastakov MY, Baer K, Xu R, Fitzsimons H, During MJ. Combined injection of rAAV with mannitol enhances gene expression in the rat brain. Mol Ther. 2001 3(2):225-32.

Stability studies carried out in-house show rAVE vectors to be highly stable at temperatures of 4^oC or less. We recommend aliquoting upon reciept and storing at -80^oC. Once an aliquot is thawed it can be stored at 4^oC for up to one month.

At room temperature, activity of the vector will reduce with time. Studies conducted indicate a drop in transgene expression in HEK293 cells of 19% after 3 days and 30% after 17 days.

What's the difference between physical and genomic particles?

rAVE vectors are supplied to a minimal concentration of 
1 x 10¹⁰ genomic particles per ml. This relates to the number of AAV particles that have been successfully packaged with the genome to be delivered. During the AAV packaging process many particles are formed lacking the genomic DNA. These lack the ability to transduce the cells they come into contact with and are therefore non-functional. As a researcher you want functional AAV particles. Therefore at GeneDetect we supply the vector product at a minimal guaranteed concentration of genomic particles. For comparison purposes with our competitors who only quote the number of physical particles they supply our physical particle titers are typically 5 x 10¹² to 1 x 10¹³ physical particles per ml.

What is the difference between genomic titer and infectious titer?

The infectious titer measures the number of vector particles that are actually infectious and this number will generally be lower than the genomic titer. Calculation of the infectious titer requires transduction of cells and transgene expression analysis (by FACSCAN analysis of protein expression for example). Given the assumption that all particles carrying the genome should be infectious, genomic titer and infectious titer should be the same for any given vector preparation. However investigators have performed comparisons and shown this is not always the case. The ratio appears to span a wide range from 1:2 to 1:200 (genomic to infectious titer) depending on the publication cited.

Do you have an MSDS for the rAVE products?

Yes. Download the rAVE MSDS  

Is there a limit to the size of the gene that can be delivered by rAVE?

We must stress that AAV has a packaging limit and that for incorporation into the full SAR-CAG promoter construct the gene must be no larger than 1600bp. If your gene is larger it may be incorporated into a smaller construct by removing some of the stabilizing elements such as WPRE or SAR (see below).

My gene of interest is slightly larger than the 1600bp limit for incorporating into your standard rAVE vector? Is there an alternative vector that can be used?

Possibly. rAAV has a packaging capacity of 4.7kb. Given the size of the promoter and the regulatory and stabilizing elements present in our standard vector, a 1600bp transgene is pretty much the limit allowed. By removing some or all of the additional elements (such as WPRE or SAR) the space available for the transgene will be larger. However these elements are beneficial and you run the risk of losing some of the vector's efficiency.

Why do you recommend the use of mannitol in delivery of rAVE to the brain?

Mannitol is a hyper-osmotic compound which has the ability to transiently disrupt the blood brain barrier following administration. As result we have found spread of an rAVE vector when co-infused with mannitol improves dramatically. Depending on your application this may be of benefit.

Question not covered by FAQ?