AVU Core Facility
Head: Lorena Zentilin
Technicians: Marina Dapas, Michela Zotti
AAV has a single-stranded DNA genome containing two genes. The Rep genes codes for the replicative proteins; the Cap genes gives rise, through alternative splicing, to 3 structural capsid proteins.
One is the Rep gene coding for the replicative proteins, and the other is Cap which gives rise by alternative splicing to 3 structural capsid proteins. To construct an AAV vector the viral genes are removed and replaced by an expression cassette containing the gene of interest. Only two short regions of the AAV genome, the Inverted Terminal Repeats (ITR) remain in the vector.
The AVU is housed in a a negative-pressured room physically separated from the surrounding laboratories by an air-lock space and is equipped with all the instrumentations needed for cell growth and vector purification.
The AVU routinely produces AAV vector stocks using an helper-free, dual plasmid transfection procedure, in which an AAV vector plasmid containing the gene of interest is co-transfected together with a packaging/helper plasmid into HEK293 cells.
The vectors can be used for research both in vitro, using appropriate target cells, or in vivo, in small experimental animals.
The AVU performs the following activities
Plasmid amplification and purification.
Vector pakaging and purification from cellular lysates by centrifugation over a CsCl gradient, followed by dialysis.
Vector titration by real-time PCR.
Functional assays (according to the specific exogenous gene and the type of application).
Characteristics of the final vector preparations
Routinely, a single rAAV stock is prepared starting from 30 150 mm-cell culture dishes.
Yield: 1×103 viral particles/cell.
Physical titer: 5×1011 to 1×1012 viral genome particles per ml.
Final volume of a standard vector batch: 3 ml in phosphate buffered saline (PBS).
The rAAV vector batches are stored at 80°C.
The AVU has availabe control AAV-GFP and AAV-LacZ vector preparations.
On specific demand, and upon proper authorization, the AVU can produce AAV vectors pseudotyped with different capsid serotypes, which may be more efficient for the transduction of certain types of target cells; serotypes 1 to 8 are currently available.
How to access the AVU
In order to access to the AVU Facility, please contact:
Dr. Lorena Zentilin