FAQ

Serotype selection: For in vitro studies, AAV-DJ and AAV6 are common choices. AAV-DJ generally infects over 80% of cultured cells, while AAV6 is particularly effective with blood-derived cells.

Gradient dilution infection: Similar to in vivo experiments, we advise conducting 3-4 AAV gradient dose infections to find the optimal transgene expression level and to ensure the dose is not cytotoxic.

Assay time: Typically, assays should be performed 2-7 days post-infection to assess the effects accurately.

Serotype selection: If you’re uncertain about the best AAV serotype for your experiments, we recommend testing at least three different serotypes in your system using our control rAAV with fluorescent reporter constructs.

Gradient dilution infection: Since transgene expression levels can vary by gene, we suggest performing 3-4 AAV gradient dose injections to identify the ideal expression level before starting formal experiments.

Experimental control: Use a GFP-positive control vector that matches the serotype and promoter of your experimental vector.

Recombinant AAV (rAAV) is a powerful tool for delivering exogenous genes, and its use in in vivo research is growing rapidly. However, the ideal injection site, rAAV quantity, concentration, and incubation period can vary widely based on the specific experiment and scientific field. Due to this variability, a universal injection protocol for AAV does not exist, so researchers need to explore each of these variables independently, guided by their unique research goals.

Serotype selection: For cells cultured in vitro, AAV-DJ and AAV6 are the most common choices. In conventional cell culture, AAV-DJ can infect more than 80% of cells, while AAV6 has the strongest infective potency against blood-derived cells.

Gradient dilution infection: The level of transgene expression driven by rAAV may vary substantially across different genes. We therefore recommend that you perform 3-4 AAV gradient dose injections to determine the ideal gene expression level for each rAAV before performing any formal experiments and to verify that your experimental dose is not cytotoxic.

Assay time: usually 2-7 days after infection.

Serotype selection: If you are unsure which AAV serotype is most suitable for your experiments, we advise that you test the infection rates of 3 or more different serotypes in your experimental system with our rAAV fluorescent reporter constructs.

Gradient dilution infection: The level of transgene expression driven by rAAV may vary substantially across different genes. We therefore recommend that you perform 3-4 AAV gradient dose injections to determine the ideal gene expression level for each rAAV before performing any formal experiments.

Experimental control: We advise the use of a GFP positive control vector of the same serotype and promoter as your experimental vector.

As an effective tool for delivering exogenous genes, rAAV are being used by a rapidly increasing number of in vivo researchers. Nevertheless, the ideal injection site, rAAV quantity, rAAV concentration, and incubation period may vary across experiments and scientific fields. Due to this variability, it is difficult to adopt a universal AAV injection protocol that is applicable for all experiments. Clients must therefore explore each of these variables independently based on their specific goals.

AAV vectors stand out for their safety, low immunogenicity, ability to transduce non-dividing cells, and potential for long-term gene expression without integrating into the host genome. These features make them particularly attractive for gene therapy applications targeting diseases where long-term expression and safety are paramount.

However, the limited packaging capacity is a constraint when delivering larger genes. In contrast, vectors like adenovirus and HSV can carry larger genetic payloads but come with higher immunogenicity and safety concerns. Lentiviral and retroviral vectors offer stable, long-term expression through genome integration but carry risks associated with insertional mutagenesis.

By leveraging the unique advantages of AAV, such as tissue-specific targeting through various serotypes and a favorable safety profile, therapies can be designed for a range of genetic disorders with minimized risks. These characteristics contribute to the growing preference for AAV vectors in both research and clinical gene therapy programs.

As of now, nine naturally occuring serotypes of human AAV have been discovered (AAV1/2/3/4/5/6/7/8/9) and widely applied in scientific research. AAV10 and AAV11 were first discovered in non-human primates in 2004, and no cross-reactivity was observed between AAV10, AAV11, and AAV2, making them promising candidate vectors. Subsequently, researchers isolated AAV12 and AAV13 from simian adenovirus, with limited research on these serotypes currently. Based on these wild-type AAVs, researchers have developed many AAV mutants, such as AAV-DJ and the PHP series, through various modification strategies.

Due to differences in the spatial structure of capsid proteins among AAV serotypes, there are significant variations in their recognition and binding to cell surface receptors, leading to tropism of different AAV serotypes for different tissues. When selecting serotypes, experimental purposes can refer to AAV serotypes used in peer-reviewed literature. For example, AAV1 and AAV9 are more commonly used in brain research than other wild-type AAV serotypes, while AAV6 exhibits higher lymphocyte selectivity.

There are also many engineered serotypes that have been modified or engineered to enhance specific properties for gene therapy applications. These modifications can include alterations to the capsid proteins to change tissue tropism, improve transduction efficiency, evade immune responses, or increase payload capacity. Engineered AAV serotypes have been developed through various strategies such as directed evolution, rational design, or hybridization of existing serotypes. These engineered serotypes offer enhanced performance and versatility, making them valuable tools for targeted gene delivery in biomedical research and therapeutic applications.

PackGene offers nearly 100 serotypes for our packaging service to assist your research work.

Additionally, the development of AAV mutant serotypes with more tissue specificity and stronger infectivity is crucial for innovation in AAV-mediated gene delivery. PackGene provides comprehensive AAV serotype engineeringg services to offer you a one-stop solution.

However, despite the tissue tropism of wild-type AAVs to some extent, the infection of non-target tissues cannot be completely avoided. In such cases, combining tissue- or cell-specific promoters with serotypes can greatly enhance AAV specificity. PackGene offers various tissue-specific promoters, such as the muscle-specific promoter MHCK7-2 and the liver-specific promoter TBG669. Our piVector Design embed in our online ordering system offers various promoters including universal and tissue specific promoters. You may easily build your vector into our AAV backbones that have been rigorously verified for effective viral packaging.