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Viral transduction

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Overview

Rapid and efficient AAV concentration

AAV vectors can be used to transduce genes into both proliferating and nonproliferating cells and can impart long-term transgene expression in nondividing cells. In addition, AAV has little immunogenicity and is suitable for the transduction of genes into whole organisms (as an in vivo transduction tool). However, it is necessary to prepare highly concentrated AAV vectors for transduction, since the amount of AAV vector injected into an individual animal depends on the administration method and target species, and the injection volume for commonly used animals (e.g., mice) is limited due to their small body size. AAVpro Concentrator provides a simple, highly efficient method for concentrating AAV particles of any serotype from cell culture supernatants using a standard centrifuge. This kit can be used for various applications, such as the concentration of AAV vectors in culture supernatant with or without serum, and buffer exchange of AAV vector solutions.

  • Efficient concentration of AAV particles from cell culture supernatant
  • Simple mix-and-spin protocol requires less than one hour of hands-on time and avoids ultracentrifugation
  • Filtration steps included for removal of impurities
  • Scalable method enables processing of various input volumes

Simple workflow  

AAVpro Concentrator allows easy and efficient concentration of AAV vectors using a standard centrifuge. In contrast with other concentration methods that involve only precipitation of AAV particles, AAVpro Concentrator incorporates filtration steps to yield AAV preps of higher purity. The scalable mix-and-spin protocol can accommodate a range of input volumes and requires less than one hour of hands-on time. Figure 1 illustrates the workflow for preparing and purifying AAV vectors using AAVpro Concentrator.

Figure 1. An outline of the AAV vector enrichment process using AAVpro Concentrator.

AAV concentration  

Concentration of AAV vectors from serum-free culture supernatant

Culture supernatant containing AAV1 vectors expressing ZsGreen1 was concentrated from a starting input of 40 ml (one T225 flask) down to a final volume of 200 μl using AAVpro Concentrator. The viral titer was measured before and after concentration to determine the viral recovery rate using the AAVpro Titration Kit (for Real Time PCR) Ver.2.

Figure 2. Concentration and recovery rate of AAV1 vectors concentrated using AAVpro Concentrator.

AAV titer  

Biological titer of concentrated AAV vectors

A biological assay commonly used to test the infectivity of an AAV preparation involves infecting permissive cells with AAV and measuring transgene expression to determine a titer in transducing units (TUs). To confirm the infectivity of AAV prepared using AAVpro Concentrator, flow cytometric analysis was used to compare ZsGreen expression in HT1080 cells infected with AAV1-ZsGreen1 vectors before or after concentration. HT1080 cells were infected with AAV at 5,000 or 500 vg/cell (MOI) and analyzed by FACS after three days. Concentrated AAV particles administered at MOIs of 5,000 or 500 were able to infect cells at rates of 44.7% or 9.2%, respectively.

Figure 3. AAV titration in transducing units, as determined by FACS analysis of ZsGreen1 expression in HT1080 cells.

Conclusions  

AAVpro Concentrator can be used to concentrate AAV particles of any serotype from cell culture supernatants, including particles prepared using any AAV system from Takara Bio. Compared to ultracentrifugation, the user-friendly AAVpro Concentrator protocol is easier to execute, requires much less hands-on time, and can be performed using standard laboratory equipment. Processing of 40 ml of culture supernatant down to a final volume of 200 µl resulted in a 95-fold increase in AAV titer at a recovery rate of ~48%. The incorporation of filtration and ultrafiltration steps in the AAVpro Concentrator protocol allows for removal of impurities that may interfere with downstream applications. AAVpro Concentrator allows for processing of supernatants with or without serum, and can also be used to perform buffer exchanges.

Related Products

Cat. # Product Size License Quantity Details
6674 AAVpro® Concentrator 1 Kit USD $407.00

AAVpro Concentrator provides a simple, highly efficient alternative to ultracentrifugation for concentrating AAV particles of any serotype from cell culture supernatant. In contrast with other concentration methods that involve only precipitation of AAV particles, AAVpro Concentrator incorporates filtration steps to yield AAV preps of higher purity. The scalable mix-and-spin protocol can accommodate a range of input volumes and requires less than one hour of hands-on time. The AAVpro Concentrator reagent is provided as a 4X concentrate, which can be applied in proportion to the amount of culture medium being processed. The kit includes reagents and filtration columns for processing up to 150 ml of culture supernatant for two samples.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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6233 AAVpro® Titration Kit (for Real Time PCR) Ver.2 100 Rxns USD $552.00

The AAVpro Titration Kit (for Real Time PCR) is a kit for determining the titer of adeno-associated virus (AAV) using real-time PCR. This kit contains all of the reagents necessary for extraction of AAV particles from AAV vector-producing cells and real-time PCR assay of virus genome. This kit allows more precise and rapid quantification than conventional DNA blotting or ELISA methods. Quantification is based on amplification of the ITRs (inverted terminal repeats) of AAV, and therefore titer measurement is possible for various serotypes of AAV.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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