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rAVE products and custom services  



rAVE Vectors

Custom
 Package only
 cDNA
 No cDNA
Reporter gene 
crAVE 
bi-rAVE 

rAVETM custom AAV2 serotype viral vectors. 

Recombinant adeno-associated viral vectors (rAAV) are quickly establishing themselves as highly versatile gene delivery agents for gene therapy1,2 and functional genomic3,4 studies. Derived from a non-pathogenic virus from the Parvoviridae family, rAAV (rAVETM) vectors can efficiently transfer genes of interest to a broad range of mammalian cell types leading to high levels of stable and long-term expression after a single application. The lack of immunogenicity and no known pathogenicity make rAAV arguably the gene therapy vector of choice for human clinical trials. 

COMING SOON. AAV mediated delivery of siRNA for in vitro and in vivo gene silencing.

1. Download a recent technical report   

2. Email for further details. Sales@genedetect.com

Publications using rAVETM gene delivery reagents have recently appeared in the Lancet, Nature, Nature Medicine, Science, Nature Genetics and PNAS. See our rAVE library for specific references.

Download some recent samples publications using GeneDetect AAV technology:

     

Continue reading below for further background information on our rAVETM product line. 

Click here to select products from our rAVETM range.

Uses for rAAV
rAAV can be used in standard laboratory settings
Production of rAVETM vectors
rAAV serotypes
rAVETM expression cassettes
rAVETM vector purity
Our production facility
rAVETM products and custom services
Pricing
Production time-lines
How supplied
rAVE FAQ (frequently asked questions)
Image Gallery
Protocols
rAVE-specific reference List

Uses for rAAV

As a gene delivery tool rAAV vectors are powerful and extremely versatile. Studied extensively for over a decade, rAAV biology is well understood with the vector having been tested and used successfully in a large range of cell types and tissue. Below is a table listing some of the tissue suitable for transduction by AAV, accompanied by references. We also list a selection of applications whereby the use of rAAV to deliver therapeutic, functional, mutated or inhibitory genes has been highly effective in both research and clinical fields.

Cell lines Tissue in vivo applications
HEK293 5,6,7,8,18 Muscle 15,16,17,18,19,20,38 Targeted gene therapy of disease
HeLa 5,6,18 Heart 21,22,29,50 cystic fibrosis 31,32
RPE 5 Brain 24,25,26,27,30,41,49,52,54 hemophilia 20,38
Rho 0 7 Liver 11,23,30 Duchenne's Muscular Disease 19,39
143B (human) 8 Lung 28,29,30,31,32 Parkinson's Disease 24,40,41
Fibroblasts 9,10 Retina 19, 33, 34, 35 Canavan Disease 25,42
COS 11 Alimentary tract 36, 37 ischemia 18,21
Primary cell cultures 10, 55 Spleen 11 rheumatoid arthritis 43
Hematopoietic cells 12,13,14 hypertension 50,51
CHO 14 Knock-outs


GFP expression (green) in brain (hippocampus) 
4 weeks
after stereotaxic injection of rAVE-GFP
vector (2
ml, 1 x 108 genomic particles).
cre-lox 44,45,46,47,48
antisense AAV 49,50,51
Generation of disease models 45, 52
Functional genomics 3,4,53
Regulatable expression systems 33,53,54
Genetic vaccines 36,55,56,57
Anterograde tracing58

rAAV can be used in standard laboratory settings

rAVE vectors in which the transgene does not encode a tumorigenic gene product or toxic molecule are assigned under NIH guidelines as Risk Group 1 organisms. By definition these agents are not associated with disease in healthy adult humans and as such require only BL1 containment status.

For rAVE vectors encoding toxic or tumorigenic transgene products Risk Group assignment will depend on the transgene. Subsequently containment requirements must be determined by the user.

Production of rAVETM vectors

With many years of rAAV vector experience, we offer the latest generation rAAV stocks optimised for high levels of transgene expression. Generation of our rAVETM vectors is achieved by a triple transfection of HEK 293 cells with 3 plasmids. The cis-acting AAV plasmid carries the rAVETM expression cassette containing the gene of interest flanked by the AAV inverted terminal repeats (ITR), while the other two plasmids contain genes encoding structural AAV capsid proteins and other adenoviral helper functions necessary for viral packaging. We then apply our modified affinity column purification methods59,60 to generate highly purified rAVETM vector stocks.

rAAV serotypes

Recombinant adeno-associated virus can exist as several different serotypes which differ physically in the composition of their capsid protein coat. To date, the majority of rAAV vector work has been based on the AAV serotype 2 (AAV2) and numerous applications of these vectors have been reported. Identification of the heparin sulfate proteoglycan61 and co-receptor fibroblast growth factor62 as receptors that mediate cellular entry for rAAV2 has led to development of rAAV purification methods based on heparin affinity chromatography. 

More recently, vectors are being developed from the AAV serotypes 1 and 5 (AAV1, AAV5), that vary in their affinities for certain cell types63,64. Unlike AAV2, the mechanisms by which other AAV serotypes enter host cells are not yet fully understood. Therefore to date no affinity matrices for purification of AAV1 or AAV5 exist. Current purification relies on cesium chloride gradients to isolate the viral particles, the resulting stocks containing a high degree of contaminating proteins. At GeneDetect we are optimizing this process, as well as looking for target receptors on which to base affinity purification of AAV1 and AAV5. In the very near future we hope to bring you these serotype options, produced to the very high standards of purity associated with our AAV2 vectors.

rAVETM expression cassettes

The secret to achieving high levels of gene expression lies in our optimized gene expression cassettes.


Above. Diagrammatic representation of a rAVE expression cassette. The rAVE cassette is flanked by the AAV inverted terminal repeats (ITR). The transgene is driven by the CAG promoter. Addition of regulatory elements SAR and WPRE enhance transgene expression. HA tags (not shown here) can also be added to either the 5 or 3 end of the gene if required. The size of the each of the elements are shown in bp. As AAV has a packaging limit of 4.7kb, the size of the transgene in this construct must be less than 1600bp (although see the FAQ). 

Left: GFP expression in HEK293 cells following transfection with rAVE expression plasmids. Addition of the SAR element improves GFP expression 4-fold. 

Why do we rAVE about our expression cassettes? Using a novel stabilized pAAV backbone, our constructs incorporate a hybrid chicken B-actin/CMV enhancer (CAG)65 promoter capable of directing very high levels of transgene expression in a wide range of cell types. Inclusion of a cis-acting woodchuck postregulatory regulatory element (WPRE)66,67 and the scaffold-attachment region (SAR)68,69 allows for increased transgene expression levels.

 

Below: FACS analysis of GFP expression in HEK293 and Neuro2A cells following transfection with plasmids containing rAVE expression cassettes.  

Expression cassettes:

A. pAM/NSE-GFP-SV40polyA
B. pAM/NSE-GFP-WPRE-SV40polyA
C. pAM/EF-GFP-WPRE-BGHpolyA
D. pAM/CMV-GFP-WPRE-BGHpolyA
E. pAM/CAG-GFP-WPRE-BGHpolyA

 

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 or there are no commercial antibodies available to my protein. Weve solved this problem for you by including options to incorporate a haemaglutinin (HA) tag sequence either to the 5 or 3 end of your gene of interest enabling easy detection and ability to distinguish endogenous from transgene expression.

rAVETM vector purity

All rAVETM vectors are purified to the highest standard using the latest innovations in AAV technology. For our AAV2 vector serotype, this involves affinity purification of the viral particles using immobilised heparan sulfate proteoglycan. 

Figure: SDS-PAGE analysis of 3 different grades of rAAV purification. a = crude, b = cesium chloride fractionated, c = affinity purified.

Following purification protocols developed and optimised in-house we can consistently generate rAVETM vectors to purity levels of 95% or better resulting in a vector product that is more efficient. Unlike crude AAV preparations, affinity purified rAAV2 does not contain transgene contaminants produced during packaging. Such contaminants within a vector stock can lead to problems of pseudo-transduction which often results in transduction misinterpretation70. By using rAVETM vector products investigators can eliminate these problems.

Our production facility

Our world-class production facility enables us to manufacture rAVETM vectors with the highest possible stringency, resulting in an extremely pure, high quality product every time. Our production facility is currently in the process of obtaining "Good Manufacturing Practice" or GMP certification, and as such adheres to a rigorous set of standard operating procedures for all stages of rAVETM vector production. This means that you, the customer, get reliable, reproducible, high purity, high titre vector stocks every time you order! In fact, vector stocks generated in our facility have already been used to treat patients in the first clinical trial in the world to use AAV in the brain25,42, and as such our stocks have been subjected to extensive independent testing. You know when you order rAVETM vectors, you are buying top quality products for your research!

Click here to take a look at our facilities.

rAVETM products and custom services

At GeneDetect we offer an extensive range of rAVETM products and services. 

Click here to select products from our rAVETM range.

We will design and manufacture rAVETM vectors optimised for your needs in consultation with you and our in-house experts. 

1. Packaging Service only. GD1000-RV

We package and affinity-column purify your vector. Simply supply your gene of interest in a pAAV vector ensuring all genetic components to be packaged are flanked by AAV2-specific ITRs.

2. Our Standard Product. GD1001-RV

Our standard rAVETM vectors are driven by a chicken -actin promoter combined with a CMV immediate early enhancer and are boosted by the addition of enhancing regulatory elements. All you need to provide is a small quantity of cDNA containing your gene and our technical staff will clone it into our pAAV backbone.

3. Reporter gene and empty vectors. GD1004-RV

As well as made-to-order rAVETM products, at GeneDetect we offer a selection of rAVETM vector expression systems for reporter genes such as GFP, luciferase and lacZ. Such genes and gene products are regularly used as positive controls in many research applications and are ideal for retrograde and anterograde tracing. 

4. COMING SOON. AAV mediated delivery of siRNA for in vitro and in vivo gene silencing. 

Please email for further details Sales@genedetect.com 

Additional services

5. We can clone your gene of interest.

If you do not have cDNA for your gene of interest we can clone it from our selection of rodent and mouse cDNA libraries. Once in the pAAV backbone, the gene is then packaged into rAAV, harvested and purified. 

6. H
aemaglutinin (HA) tagging service.

Unlike other suppliers of AAV vectors we can offer an optional gene tagging service that allows for accurate tracking of your gene product in tissue high in endogenous gene levels.

Prior to shipping all rAVETM vectors are tested for purity, and are titered to ensure the sample contains no less than 1 x 1010 genomic particles per ml.

Pricing

Catalog Number Service Cost (USD)

GD1000-RV Packaging service, customer supplies gene in pAAV vector with AAV2-specific ITRs $3495
GD1001-RV Customer supplies cDNA that is cloned into our expression cassette before AAV is prepared. $4490
GD1004-RV Ready made reporter gene vectors 
(GFP, luciferase, LacZ, empty)
$2995

Optional Clone Rat or Mouse gene for incorporation into AAV $6995
Optional Addition of Haemaglutinin tag to either 5' or 3' $1295

Production time-lines

The following production time-lines are estimates and represent "working days". Typically, turn-around will be within 5-6 weeks of receiving your DNA and will depend on the production options you specify.

1. Cloning of gene into AAV, 6 days
2. DNA amplification (construct and helpers), 2 days
3. AAV packaging, 7 days
4. Purification and dialysis, 3 days
5. Titer and quality control, 2 days
6. Addition of HA tag, 10 days
7. Cloning of customers gene, 10 days to 1 month.

How supplied

rAVE vectors are supplied in 0.3 ml (300l) of phosphate buffered saline (PBS) containing 1mM MgSO4

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. 

How does the genomic particle titer relate to the infectious titer? See here.

As a researcher you want functional AAV particles. 

Used at 1:100 in media, rAVE will infect and express transgene in 30-75% of the treated cells within 3 days, depending on cell type and transgene being used.

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.

 rAVE guaranteed minimum titer supplied:
 (0.3ml) > 1 x 1010 genomic particles per ml

 (our typical titer range ~  1 x 1012  to  5 x 1012 GP/ml)

 MINIMUM guaranteed titer typically equates to: 
 (0.3ml) > 5 x 1012 to 1 x 1013 physical particles per ml

rAVE vectors are shipped at 0-4C in a Deep Chill Shipper and are delivered door-to-door by Fedex. Click here for more information.

 

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