Powerful Gene Delivery and Expression System

Stratagene’s AAV Helper-Free System* allows the high-efficiency gene transfer of viral-based systems with increased safety over other viral-based systems. These important characteristics have led to increased interest in AAV vectors for many applications including gene therapy¹ and drug discovery. Recombinant AAV-2 has a broad host range, infects both dividing and non-dividing cells, and integrates into the host cell for long-term, stable expression.

Unmatched Safety

AAV is naturally replication-deficient and normally requires co-infection with an unrelated helper virus, such as adenovirus, in order to replicate. However, Stratagene’s AAV Helper-Free System completely eliminates the need for helper virus, so gene delivery and expression occur with unparalleled safety. The system is composed of the minimum number of adenoviral gene products required for helper-free transient production of virus and is based on the system developed at Avigen, Inc. by Peter Colosi and colleagues². The components that contain the necessary genes for generating infectious virions are positioned on separate plasmids, and because these plasmids do not share regions of homology, they cannot recombine to form wild-type virus. Eliminating the need for helper virus³—coupled with the fact that AAV virus has never been associated with any known human disease gives this system a high biosafety profile.

Three-Step Procedure

The expression of genes of interest with the AAV Helper-Free System consists of three steps: 1) transfection of a packaging cell line, 2) collection of high-titer recombinant virus from the cell lysates, and 3) transduction of target cells for expression studies.

Novel Helper-Free Titer Protocol

To determine the titer (i.e., the number of infectious viral particles per unit volume) of virus preparations, a reporter virus is produced in parallel in this system. We have developed a novel three-day tittering method** that eliminates the need for co-infection with adenovirus. Traditionally, co-infection with adenovirus shortened the time required to generate titers from ten to three days. Our new helper free method involves applying chemical reagents known to enhance transduction efficiency and increase expression from the CMV promoter***. Our novel chemical induction method eliminates the need for co-infection with adenovirus and allows titers to be obtained in two to three days.

Now you can deliver your gene to a broader range of hosts with extremely high efficiency for long-term, stable gene expression using a safe, effective viral-based system.

REFERENCES

1. Kay, M. A., et al. (2000) Nature Genetics 24: 257-2612.

2. Matsushita, T., et al. (1998) Gene Therapy 5: 938-945.

The gene of interest is inserted into the pAAV-MCS expression vector (Figure 1). Alternatively, if you are interested in manipulating your gene of interest, for example by mutagenesis, before expression, you may insert your gene of interest into the pCMV-MCS shuttle vector for any manipulation and then transfer your gene to the pAAV-LacZ expression vector for expression studies. In step one, the recombinant construct is cotransfected into a packaging cell line with pAAV-RC and pHelper plasmids, which each encode specific AAV gene products necessary for inducing the lytic phase and packaging virions. In step two, replication deficient, recombinant AAV virions are collected from cotransfection lysates and used for step three: transduction of target cells for expression studies (Figure 2). Infection conditions that are nonpermissive for replication prompt the gene of interest to integrate into the genome of target cells and establish stable expression.

* See license reference 4 on page 32
** Patents pending
*** See license reference 6 on page 32
**** Patents pending
See license reference 8 on page 32

go to product page download Adobe PDF document trademarks and licensing