Adenoviral-Associated Viral Vector Services
Adenoviral associated virus (AAV) is a simple, non-pathogenic, single-stranded DNA (ssDNA) virus requiring a helper virus, usually an adenovirus, for replication. It encodes two genes, cap and rep, sandwiched between inverted terminal repeats (ITR). The ITRs both define the beginning and the end of the viral genome and contain the packaging sequence. The cap gene encodes viral capsid proteins, and the rep gene product is involved in viral replication and integration. AAV infects a variety of cell types. In the presence of the rep gene product, the viral DNA can preferentially integrate into human chromosome 19.
To generate an AAV vector, the rep and cap genes are replaced with a transgene. The size of the transgene is limited to the length of the AAV genome, 4.7 kb. Production of the recombinant AAV vector requires that rep and cap be provided in trans along with the helper virus. The current method of AAV preparation is to co-transfect two shuttle vectors into cells already infected with helper adenovirus: one shuttle vector contains the viral vector and the second is for rep and cap expression. Although this method is cumbersome, prone to adenovirus and wildtype AAV contamination, and limited to low virus accumulation, its host genome integration strategy and sustained gene expression are useful to many studies.
Services (SC1167):
- Recombinant AAV vector construction: Cloning plasmids are available to express the transgene as single gene product or simultaneously with GFP reporter under control of IRES for transfection efficiency analysis. The transgene can be driven by either internal promoters, such as CMV, PGK, Ubc, MCK, Desmin, or a synthetic muscle-specific promoter.
- Recombinant AAV production: Recombinant AAV is generated in 293A cells by co-transfection with three plasmids, an AAV rep/cap expression plasmid, an adenovirus miniplasmid containing the required helper function, and the AAV viral vector plasmid. This method is highly flexible for the generation of AAV viral vectors with a variety of different AAV capsids, including those derived from AAV serotypes 1, 2, 5, and 6, which differ in host ranges.
- Recombinant AAV preps: Recombinant AAV particles are isolated from the transfected cell lysates either by heparin column chromatography (for AAV serotypes 2 and 5) or iodixanol density gradients (for all other serotypes). The final virus preps are dialyzed against PBS and titered by dot blot hybridization. The infectious titers are determined before final release.
- Recombinant AAV characteristics: Routinely, a single recombinant AAV stock is prepared starting from 30×150 mm-cell culture dishes.
- Yield: 1×103 viral particles/cell
- Biological titer: 108-109 pfu/ml for concentrated
- Final volume: 1 ml of purified virus in phosphate buffered saline (PBS)
- Storage: The recombinant AAV vector batches are best stored at -80°C
Additional Services:
GenScript offers AAV-GFP and AAV-LacZ vector preparations that can be used as controls. Upon request, GenScript can produce customized AAV vectors pseudotyped with different capsid serotypes.
 For quotation requests, please download and complete GenScript Viral Vector Services Quotation Request Form, and email or fax it to us with a detailed description of project requirements. For questions, please use our secure web server. However, you may also contact us by email at order@genscript.com, phone, or fax.
Orders can be placed by phone, email, or fax with either a formal PO (Purchase Order) or credit card. Please email or fax us a completed GenScript Viral Vector Services Quotation Request Form. Our customer service representatives are available 24 hours Monday through Friday. If target gene is physically available, please submit your target gene along with a hard copy of completed GenScript Viral Vector Services Quotation Request Form to Viral Vector Services, GenScript Corp., 120 Centennial Ave., Suite 105, Piscataway, NJ 08854, US.
 |
Order by email: order@genscript.com |
 |
Order by phone: 1-877-436-7274 (Toll-Free) 1-732-885-9188 |
 |
Order by fax: 1-732-210-0262 1-732-885-5878 |
|
