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viral vector icon Adenoviral Guide

Adenoviral Components

The adenovirus genome is a linear, 36-Kb double-stranded DNA (dsDNA) molecule containing multiple, heavily spliced transcripts. At either end of the genome are inverted terminal repeats (ITRs). Genes are divided into early (E1-4) and late (L1-5) transcripts. There are 57 accepted human adenovirus types, and most adenoviral vectors are based on Ad5. Ad5-based vectors use the Coxsackie-Adenovirus Receptor (CAR) to enter cells.

Recombinant adenovirus has two genes deleted: E1 and E3. E1 is supplied by the adenovirus packaging lines 293 or 911; its deletion from the viral vector renders the virus replication incompetent. E3 is involved in evading host immunity and is not essential for virus production. Deletion of these two components results in a transgene packaging capacity of >8 Kb. Constructs contain left and right arms to facilitate homologous recombination of the transgene into the adenoviral plasmid.

Please see our Biosafety guide for more information on working with adenovirus.

adenoviral plasmid structure cartoon
Overview of Adenoviral Plasmid System

AdEasy™ for Adenoviral Vector Construction

AdEasy™, developed by Bert Vogelstein , is by far the most popular method for creating adenoviral vector constructs. The system consists of two types of plasmids: shuttle (or transfer) vectors and adenoviral vectors. Find vectors for this system on our adenovirus plasmid page.

The transgene of interest is cloned into the shuttle vector, verified, and linearized with the restriction enzyme PmeI. This construct is then transformed into AdEasier-1 cells , which are BJ5183 E. coli cells containing pAdEasy™. pAdEasy™ is a ∼33Kb adenoviral plasmid containing the adenoviral genes necessary for virus production. The shuttle vector and the adenoviral plasmid have matching left and right homology arms which facilitate homologous recombination of the transgene into the adenoviral plasmid. One can also co-transform standard BJ5183 with supercoiled pAdEasy™ and the shuttle vector, but this method results in a higher background of non-recombinant adenoviral plasmids.

Recombinant adenoviral plasmids are then verified for size and proper restriction digest patterns to determine that the transgene has been inserted into the adenoviral plasmid, and that other patterns of recombination have not occurred. Once verified, the recombinant plasmid is linearized with PacI to create a linear dsDNA construct flanked by ITRs. 293 or 911 cells are transfected with the linearized construct, and virus can be harvested about 7-10 days later.

Vogelstein designed multiple shuttle vectors for different purposes. The pAdTrack series contains an IRES-GFP construct that enables co-expression of GFP with the transgene of interest. With these plasmids, one can track the infection of 293/911 cells throughout virus production. During experiments, GFP can be used to sort cells infected with adenovirus, or to verify that the infection rates are equivalent across multiple viruses.

The adenoviral backbone pAdEasy-1 is suitable for most purposes. For especially long transgenes, the use of pAdEasy-2 can increase the capacity of the adenoviral vector. pAdEasy-2 does not contain the viral gene E4, adding 2.7 Kb of packaging space. However, these constructs must be transfected into 911E4 cells for virus production, as 293 cells do not contain E4.

Packaging diagram for adenovirus
Adenovirus Packaging Methods

Frequently Asked Questions (FAQ) about Adenoviral Vectors

Are adenovirus and AAV different?

Yes! AAV is a small single-stranded DNA parvovirus and was discovered as a contaminant of adenovirus preparations, whereas adenoviruses are a class of medium-sized, non-enveloped double-stranded DNA viruses. They are not related; however, AAV requires the presence of adenoviral genes E1, E4, E2a and VA for replication.

For more information about AAV, read our AAV guide .

Can I make a stable cell line with adenovirus?

No, adenoviral vectors can only be used transiently.

Are adenoviruses replication deficient?

Typically yes, because the early genes necessary for replication have been deleted from the shuttle vector. Early gene E1 is provided by the transfected cell line (either 293 or 911). To avoid creating replication competent virus, you should not serially propagate your virus as the chance of recombination events creating replication-competent virus increases with each round of amplification.

What is RCA?

RCA stands for r eplication c ompetent a denovirus. This may occur when a crossover event between your adenoviral vector and the early genes found in the packaging cell lines creates a wildtype adenovirus. The probability of a crossover event increases with each round of amplification.

Where can I find information about using the AdEasy™ System?

The laboratory of Bert Vogelstein has published their method for generation of recombinant adenoviruses: A Practical Guide for using the AdEasy™ System (127 KB) .


Packaging Vocabulary
Term Definition
pAdEasy-1 Adenovirus packaging plasmid that lacks E1 and E3
pAdEasy-2 Adenovirus packaging plasmid that lacks E1, E3, and E4; Useful for very large inserts
AdEasier-1 cells BJ5183 E. coli cells containing the pAdEasy-1 packaging plasmid
911E4 cells Cell line compatible with the pAdEasy-2 packaging plasmid; Supplies viral protein E4
E1 Supplied in trans by 293 or 911 packaging cells; Its deletion renders the virus replication incompetent
E3 Non-essential gene involved in immune evasion, deleted to increase packaging capacity
E4 Essential for viral transcription; Supplied by the pAdEasy-1 packaging plasmid or 911E4 packaging cell line
Transfer Vocabulary
Term Definition
ITR Inverted terminal repeat; Contains elements that promote bi-directional transcription
Ad5-based vector Most common type of adenoviral vector
pAdTrack Class of transfer vectors that contain IRES-GFP
pShuttle Class of transfer vectors that do not contain GFP
Additional Vocabulary
Term Definition
Coxsackie-Adenovirus Receptor (CAR) Receptor used by Ad5-based vectors to enter cells