to titer my AAV preps I perform qPCR. I have a reference plasmid for standard curve and after digesting the capsid protein i can quantify the infectious titer.

Hope it could help!

I perform qPCR as well, and ELISA, but we would like to have a 3rd assay to mesure the transduction units to be sure that our virus is infectious.

Hi Melanie, which AAV serotype are you working with? It is dificult to recommend any host cell type without knowing.

You may be well aware, but perhaps you also need to consider that the functional titer you get from a biological assay represents and strickly dependent on two factors: the actual titer of infectious particles and the sensitivity of the cell line. It can be used to normalize between virus batches, but titer on cell type A won`t tell you much about virus load on cell type B.

qPCR or ELISA-based titration estimates the amount of assembled virus particles and the only variable reminas uncontrolled is the ratio of infectious particles per total amount, which is more or less constant when following same virus production protocols. Why not settle with that?

Maybe you could inject mice with your AAV. If your transgene is supposed to be secreted then you can collect the serum and perform ELISA! or collect the transduced organ if not secreted and perform qPCR.

Thanks everyone for your replies.

Balasz : I am using the serotype AAV2. The project I'm working on is part of a grant with another lab, initially we were suppose to only perform qPCR and ELISA but we decided that transduction assay could add more accurate data.

Lea : in vivo trials are not part of this project I'm afraid. I was hoping that someone already performed a transduction assay with AAV and succeeded to have good data with it. 

Melanie, AAV2 is mostly used in the CNS (e.g. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2367141/), perhaps you should try some neuronal/neuroblast cell type instead of Hek and Hela.

You may like to try out our transduction protocol. It works great and you are welcome to try out our AAV vectors as well.

Hi, Mel

Did you get your problem solved? I met the exact same issue here. No transduction for infectious titering. Could you tell me the solution if you have solved it? Thanks

Hi Yongchang,

We stopped trying to perform in vitro transductions with AAV2. It seems that only in vivo transduction is working with AAV type 2.

Dear Esteemed Colleague,

I hope this message finds you well. Your inquiry regarding the experience with transduction assays using Adeno-Associated Virus (AAV) is highly relevant to the fields of gene therapy and molecular biology. AAV vectors are widely recognized for their efficacy in delivering genetic material to a variety of cell types, both in vitro and in vivo, with a notable safety profile. Herein, I outline key considerations and practical advice based on extensive experience in the field, aiming to support your endeavors in conducting successful AAV transduction assays.

1. Selection of AAV Serotype:One of the initial steps in planning your AAV transduction assay involves the selection of the appropriate AAV serotype. Different AAV serotypes exhibit varying tropisms for different cell types and tissues. It is crucial to choose a serotype that efficiently targets the specific cells of interest in your study. Common serotypes include AAV2, AAV5, AAV6, and AAV9, among others, each with its unique profile for transduction efficiency and tissue specificity.

2. Multiplicity of Infection (MOI):Determining the optimal MOI, which is the ratio of viral particles to target cells, is critical for achieving effective transduction while minimizing cytotoxicity. The MOI may vary depending on the cell type, the AAV serotype, and the experimental objectives. Pilot experiments to optimize the MOI for your specific system are recommended.

3. Preparation and Purification of AAV Vectors:The quality and purity of AAV vectors significantly affect transduction efficiency. It is essential to use well-characterized, high-titer AAV preparations. Techniques such as ultracentrifugation-based purification or chromatography can be employed to achieve high-purity viral stocks. Quantification of viral particles using methods such as qPCR or ELISA is necessary to determine the vector dosage accurately.

4. Transduction Protocol:For in vitro assays, cells should be seeded at an appropriate density before transduction to ensure optimal growth conditions. The addition of AAV vectors to the culture medium, possibly in the presence of adjuvants that enhance transduction (e.g., polybrene), is followed by incubation under suitable conditions. It is vital to monitor the cells for any signs of toxicity and to optimize the duration of transduction to maximize gene expression.

5. Assessment of Transduction Efficiency:After transduction, evaluating the efficiency of gene delivery and expression is crucial. This can be accomplished through various methods, such as fluorescence microscopy for GFP or other reporter genes, flow cytometry, or qPCR for quantifying vector genomes. The choice of assessment method depends on the nature of the transgene and the specific aims of the experiment.

6. Considerations for In Vivo Applications:When conducting in vivo AAV transduction assays, considerations such as the route of administration (e.g., systemic injection, direct tissue injection), the volume of injection, and potential immune responses become paramount. Proper planning and ethical approval for animal use are essential components of in vivo studies.

In summary, AAV-mediated transduction assays are a powerful tool in gene therapy research, offering the potential to elucidate gene function, model diseases, and develop therapeutic strategies. Attention to the selection of AAV serotypes, optimization of MOI, preparation of high-quality viral vectors, and careful execution of transduction protocols will significantly contribute to the success of your experiments.

Should you have further questions or require specific details on any aspect of AAV-mediated transduction, please feel free to reach out. Your success in this venture is of great interest, and I am here to offer support.

Best regards,

Check out this protocol list; it might provide additional insights for resolving the issue.