Adenovirus Packaging Protocol for Adenovirus Transfection
BioInnovatise Viral Vector Team
Updated November 18, 2024
What Is an Adenovirus Packaging Protocol and Which Is Right for My Research?
Adenovirus packaging protocols outline the steps to create replication-deficient (an important characteristic) adenoviruses to deliver genes of interest to target cells for research and or therapeutic purposes. Adenoviral packaging involves designing and cloning adenoviral vectors, recombination, linearization, packaging, amplification, and finally purification and titration.
Transfection protocols involve delivering recombinant adenoviruses to target cells for gene expression studies. These steps include: adenovirus preparation, cell preparation, cell infection, incubation, and gene expression.
This article will primarily focus on the adenovirus packaging protocol. Depending on your research application, you need to pick a specific adenovirus packaging protocol that matches your application best.
What Are The Major Differences Between Adenovirus Packaging Protocols?
Generally speaking, adenovirus packaging protocols have similar flows that start from vector design and finish with titration. However, there are differences among packaging protocols due to several factors which require the protocol to have different required materials, lab equipment, or a different series of steps in each major section of the adenovirus packaging process. Here are some of the major reasons why adenovirus packaging protocols vary:c
- Adenovirus Type:
- Certain adenovirus have tissue specifity (i.e. serotypes) which may require certain regions of the genome to be removed (frequently E1 and E5) which affects packaging efficiency. For example, Ad5 is highly efficient for packaging while Ad26 or Ad35 require optimization for large scale productions such as tailored cell culture conditions and enhancers for viral yield.
- Lentiviruses (see lentivirus packaging protocol for specific lentivirus packaging details)
- Insert Size
- For larger inserts, helper-dependent vectors or modified approaches may be used, which require different recombination and production methods.
- Learn more about various adenovirus packaging capacity limits.
- Recombination Method
- Some protocols use recombination in E. coli for faster and more efficient generation of recombinant adenoviral DNA.
- Other methods rely on direct recombination in HEK293T cells, which can be more time-consuming but avoids bacterial intermediates.
- Transfection Method
- Different reagents and techniques affect the transfection efficiency of the adenoviral DNA into packaging cells like HEK293.
- Cell Line Used
- Most protocols use HEK293 cells because they provide the E1 region necessary for adenovirus replication. Our lab prefers HEK293T cell line for it’s packaging efficiency.
- Purification Method
- Traditional ultracentrifugation is precise but labor-intensive. Our lab uses ultracentrifugation for high titer adenoviral productions.
Our adenoviral packaging team will always use the specific transfection protocol requested by the customer. If you are unsure which adenovirus transfection protocol is right for you, contact our team. The below transfection protocol is a general protocol for educational purposes and should not be used outside of BioInnovatise productions. It is specifically for helper-dependent Ad5 adenovirus.
Learn about our quick turnaround adenovirus packaging service.
The above diagram illustrates the adenovirus packaging process at BioInnovatise.
Materials Required
- Helper-dependent adenovirus DNA
- Plasmid with the required gene of interest flanked by the adenovirus inverted terminal repeats (ITRs) and a packaging signal.
- Helper virus
- Packaging cell line
- Transfection reagent
- Culture medium
- Buffers
- Reagents for titration
Protocol Procedure
- Linearize HD-Ad DNA
- Digest the HD-Ad plasmid with a restriction enzyme to linearize it.
- Purify the linear DNA using a DNA purification kit.
- Transfection into HEK293-Cre Cells
- Plate HEK293-Cre cells at 70–80% confluency in a 10-cm dish (~2 × 10⁶ cells).
- Prepare the transfection mix:
- Mix 10–20 µg of linearized HD-Ad DNA with transfection reagent (e.g., PEI at a 1:3 DNA
ratio). - Incubate for 20 minutes at room temperature.
- Mix 10–20 µg of linearized HD-Ad DNA with transfection reagent (e.g., PEI at a 1:3 DNA
- Add the transfection mix dropwise to the cells.
- Incubate cells at 37°C in 5% CO₂ for 6–8 hours, then replace with fresh medium.
- Infection with Helper Virus
- After transfection (12–24 hours), infect cells with the E1-deleted Ad5 helper virus at a low multiplicity of infection (MOI = 1).
- Incubate for 48–72 hours, monitoring for cytopathic effect (CPE) under a microscope.
- Harvesting adenovirus
- Once 90% of the cells show CPE, collect the cells and medium.
- Perform three freeze-thaw cycles to lyse cells and release adenoviruses.
- Centrifuge the lysate at 3,000 × g for 10 minutes to remove cell debris.
- Filter the supernatant through a 0.45 µm syringe filter.
- Amplification
- Use the filtered lysate to infect a fresh plate of HEK293-Cre cells.
- Repeat the infection and harvest steps to produce a higher titer of adenovirus.
- Read more about adenovirus packaging and amplification.
- Ultracentrifugation purification
- Layer the viral lysate onto a CsCl gradient and ultracentrifuge at 35,000 rpm for 2 hours.
- Extract the virus band, dilute with PBS, and re-ultracentrifuge for final purification.
- Titration
- Determine the viral titer using RT-qPCR
- Quality control assays
- Check for helper virus contamination by testing for Cre-dependent excision of the helper virus packaging signal.
- Test endotoxin levels if required for downstream applications.
Notes from the BioInnovatise laboratory:
- QC assays: Our production labs recommend A260/A280 ratio, flow cytometry, western blot/ELISA assays for adenovirus packaging.
- Safety: Handle adenovirus under biosafety level 2 (BSL-2) containment.
Want to learn more about the latest in adenoviral based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News are always collecting and publishing the latest information on adenovirus based research.
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