AAV Helper-Free System

The AAV helper-free system eliminates the requirement for wild-type adenovirus co-infection from both AAV vector production and AAV stock titering steps.

Using an AAV helper plasmid makes this adeno-associated virus system safer, and more convenient.
The AAV helper-free system features versatility in host range, high titer virus production , and long-term gene transfer potential.

The AAV packaging protocol works with a broad range of hosts and infects both dividing and nondividing cells.


Life Science – Molecular Biology – AAV Helper-Free System


  • Stable gene expression
  • High titer virus production
  • Lower toxicity compared to nonviral methods of gene delivery
  • Accomodates inserts up to 3kb
  • By eliminating the requirement for live helper virus, the AAV Helper-Free System provides a safer, purer and more convenient alternative to retroviral and adenoviral gene delivery systems. In the AAV Helper-Free system, the AAV-2 ITR-containing plasmids (pAAV-MCS, pAAV-LacZ and pAAV-hrGFP and pAAV-IRES-hrGFP) do not share any regions of homology with the rep/cap-gene containing plasmid (pAAV-RC), preventing the production of wild-type AAV-2 through recombination. To ensure that this lack of homology is maintained, it is important to use all of the components provided with the AAV Helper-Free system together.
  • The pAAV-hrGFP vector is used as a positive control in the AAV Helper-Free System.
  • This control vector uses humanized Renilla GFP (hrGFP) reporter gene for easy detection of expression in vivo using fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis, or fluorometry. pAAV-hrGFP can be used to qualitatively assess the transfection efficiency of the producer cell line and to determine the viral titer using fluorescence microscopy or by fluorescence activated cell sorting (FACS).
  • AAV-293 cells are an HEK293 cell line optimized for packaging of AAV virions in the helper-free system. AAV-293 cells, like HEK293 cells, produce E1 in trans. AAV-293 cells allow production of infectious virus particles when transfected with E1-deleted adenovirus vectors or co-transfected with the three AAV helper-free system plasmids.
  • These plasmids supply all of the trans-acting factors required for AAV replication and packaging in AAV-293 cells. Recombinant AAV-2 viral particles prepared from infected AAV-293 cells may then be used to infect a variety of mammalian cells.
  • AAV-HT1080 Cells, a human fibrosarcoma cell line, is recommended for tittering adeno-associated recombinant virus stocks for the AAV Helper-Free system. The AAV-HT1080 Cells are found to be more permissible to AAV infection than other cells lines and thus able to give more accurate viral titers. In addition, viral titer measurement protocols using AAV-HT1080 cells have been optimized and are provided in pAAV-LacZ and pAAV-hrGFP Control Applications.
Catalog no.Description
240071AAV Helper-Free System
240073AAV-293 Cells
240074pAAV-hrGFP Vector
240075pAAV-IRES-hrGFP Vector
240109AAV-HT1080 Cells