rAVE Frequently Asked Questions.
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 4şC. 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.
Vacuum insulated panel (VIP) technology (by Dow Chemical Corporation, Midland, MI) is the same technology used in thermos flasks to prevent heat dissipation. Until recently however it was not possible to form a square vacuum panel of the type needed for a shipping box since once air is removed from the panel to form the vacuum, the pressure becomes so great at the corners of the "panel" that the walls collapse. Recently Dow has overcome this issue and is now supplying the Instill VIP core (patent pending).
Deep Chill™ Shipper
GeneDetect uses the new Deep Chill™ Shipper available from PolyFoam Packers Corporation (www.polyfoam.com) to ship your products to you (and of course you get to keep the shipper and re-use it for your own needs). The Deep Chill™ Shipper is a total shipping system that utilizes vacuum insulation panel (VIP) technology to achieve a system insulation value of up to R-36.
This provides increased capacity and extended shipping time without adding additional shipping weight. The Deep Chill™ System combines vacuum insulation panels and custom molded EPS.
This unique combination is designed to offer the ultimate in product protection and reach. Each component provides a specific layer of protection.
Four components of the Deep Chill™ Shipper System
A. 1/8" inner corrugated liner-Keeps payload from damaging the Vacuum Insulation Panel without sacrificing internal capacity.
B. 1" wall vacuum insulation panel-provides an average insulation value of R-30 per inch.
C. 1 and 1/2" wall of custom molded EPS-protects the vacuum panel from the rigors of shipping, provides the first layer of thermal protection and provides an average insulation value of R-4 per inch.
D. Outer corrugated shipper-provides ample space for shipping labels and documents.
GeneDetect has extensively tested the Deep Chill™
Shipper System. Using our packaging methods we are able to maintain rAVE samples
at a temperature of 0-1şC over 7 days!!
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 4oC or less. We recommend aliquoting upon reciept and storing at -80oC. Once an aliquot is thawed it can be stored at 4oC 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
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.
Yes. Download the rAVE MSDS
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