(ATCC® VR-1616)

Product Format: frozen

Permits and Restrictions

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Strain Recombinant Adeno-associated Virus 2 Reference Standard Stock (rAAV2 RSS)
Biosafety Level 2

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Isolation
While the final rAAV2 RSS preparation was found to be pure and free of adventitious agents, the cell harvest from which it was made tested positive for Mycoplasma arginini. The rAAV2 RSS has therefore been exposed to mycoplasma, but the purified rAAV2 RSS was found by extensive testing to be free of viable mycoplasma and mycoplasma DNA. The AAVRSWG has recommended distribution of the purified reference material because the purification process includes several steps likely to be inactivating for mycoplasma. Since the material is not intended for human use, the exposure to mycoplasma does not preclude its use for control and comparison of in vitro tests.
Product Format frozen
Storage Conditions -70°C or colder
Intended Use
Recommended Use of the Recombinant Adeno-associated Virus 2 Reference Standard Stock
The rAAV2 RSS is intended for use as a benchmarking tool to qualify and validate "in house" reference materials and assays used in basic, pre-clinical and clinical research employing recombinant adeno-associated virus 2 vectors, to support comparison of data across laboratories. Due to the wide variety of methods applied to vector characterization, and even the variability of data between laboratories using the same method, correlation of results from different sources is difficult, clouding interpretation of aggregated information. It is anticipated that introduction of a common Reference Material to characterize calibration reagents and to bridge assays will facilitate comparison. Each laboratory should characterize and qualify its own "in house" reference material using the rAAV2 RSS. Extensive validation work should be performed in each laboratory using the laboratory's qualified reference material, as the availability of the rAAV2 RSS is limited.

The FDA recommends use of the rAAV2 RSS as a common Reference Material as described. This recommendation does not imply any intent to standardize assay methods across the field or to require that the values assigned to the rAAV2 RSS be duplicated during validation studies.
Comments
While the final rAAV2 RSS preparation was found to be pure and free of adventitious agents, the cell harvest from which it was made tested positive for Mycoplasma arginini. The rAAV2 RSS has therefore been exposed to mycoplasma, but the purified rAAV2 RSS was found by extensive testing to be free of viable mycoplasma and mycoplasma DNA. The AAVRSWG has recommended distribution of the purified reference material because the purification process includes several steps likely to be inactivating for mycoplasma. Since the material is not intended for human use, the exposure to mycoplasma does not preclude its use for control and comparison of in vitro tests.
Recommended Host
Recommended Vectors and Host Cells
Plasmid pTR-UF-11 [3], the recombinant AAV genome, contains the coding sequence for humanized GFP under the control of the synthetic CBA promoter and the SV40 polyadenylation signal, followed by the neomycin-resistance gene under the control of the mutant polyoma virus enhancer/promoter (PYF441) and the human bovine growth hormone poly(A) site, flanked by AAV2 TRs. The HEK293 cell line was co-transfecteed with vector plasmid pTR-UF-11 and the helper plasmid pDG-KanR (6) to generate the rAAV2-RSS. pTR-UF-11 is available from ATCC, distributed as product number MBA-331 .

The cell line used for titration of infectivity of the rAAV2 RSS, HeLa RC32 [4], is a HeLa derivative which expresses the rep and cap genes for recombinant adeno-associated virus type 2 (rAAV-2). This cell line is distributed by ATCC as product number CRL-2972 .
Growth Conditions
Temperature: 37°C
Cryopreservation
Recommended Storage and Handling Information
Vials are shipped from ATCC on sufficient dry ice to maintain the product in frozen condition until received by the end user. Immediately upon receipt, store vials frozen at -70°C to -90°C.

For use in assays of infectivity (e. g. TCID50 or GFP transduction assays), thaw vial(s) at room temperature while mixing gently. DO NOT VORTEX THE rAAV2 RSS. Keep thawed vial(s) on wet ice until use. Conduct infectivity assay(s) within 1 hour of thawing. Store the remainder of the thawed vial at 4°C and mix gently immediately prior to use.

Conduct physical assays (e. g. SDS-PAGE, capsid ELISA, or vector genome PCR assays) within 5 days of vial thaw.
Name of Depositor Adeno-associated Virus Reference Standard Working Group (AAVRSWG)
References

Moullier P, Snyder, RO. International efforts for recombinant adeno-associated viral vector reference standards. Mol. Ther. 16:1185-1188, 2008.

Potter M, et al. Manufacture and stability study of the recombinant adeno-associated virus serotype 2 vector reference standard. BioProcessing Journal 7:8-14, 2008.

Burger C. et al. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Molecular Therapy : 10(2):302-17, 2004 PubMed: 15294177

Flotte TR, e al. Utility of a recombinant adeno-associated viral vector reference standard. BioProcessing J. 1: 75, 2002.

Chadeuf G, et al. Efficient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome. J. Gene Med. 2(4):260-268, 2000. PubMed: 10953917.

Grimm D, et al. Novel tools for production and purification of recombinant adeno-associated virus vectors. Human Gene Therapy 9: 2745-2760, 1998.

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation