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 rAVE Vectors Custom Package only cDNA No cDNA Reporter gene crAVE bi-rAVE |
rAVETM custom AAV2 serotype
viral vectors.
2. Email for further details. Sales@genedetect.com
Click here to select products from our rAVETM range.
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.
rAAV can be used in standard laboratory settings
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. 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. The secret to achieving high levels of gene expression lies in our optimized gene expression cassettes.
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.
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. We’ve 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. 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 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. 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 ß-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 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. 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.
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. rAVE vectors are supplied in 0.3 ml (300µl)
of phosphate buffered saline (PBS) containing 1mM MgSO4. How does the genomic particle titer relate to
the infectious titer? See here. Therefore at GeneDetect we supply the vector product
at a minimal guaranteed concentration of genomic particles.
rAVE vectors are shipped at 0-4°C in a Deep Chill™ Shipper and are delivered door-to-door by Fedex. Click here for more information.
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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 
AAV2 vector serotype, this involves affinity purification of the viral particles using immobilised heparan sulfate proteoglycan.