Dual Expressers in RAPAd® Adenovirus Production

Two genes - one vector!


Dual expressers are adenovirus vectors that contain modifications in both the E1 and E3 regions of the genome

These modifications are designed to allow multi-gene expression from a single vector. Independent promoters provide high levels of gene expression without the complexity or inefficiency of an IRES sequence.

E1 Region: In the RAPAd® system we can use any E1 shuttle plasmid in conjunction with any E3 backbone. Making your virus with a second gene may be a transfection away!  

E3 Region: We use the E3 region to engineer common genes used throughout an experiment. This would be the common denominator among a group of related viruses. For example, you have 4 reporters you want to express in concert with CD40L, we would clone CD40L into the backbone and the reporters in the E1 shuttle plasmids. 5 clones, 4 recombinations, 4 viruses – 3 to 6 weeks.

Our dual expresser system makes it easy!

We can make your novel adenovirus with a reporter gene in the E3 region of the backbone. Our method separates the expression cassettes by 20,000 base pairs and is more stable than expression cassettes placed in tandem in the E1 region.

We can also engineer your second gene of choice into the E3 region. This eliminates the need for dual infection and guarantees that each cell receives both expression cassettes.

As seen on our RAPAd® page, we offer several E3 backbones with additional E3 deletions and reporter gene inserts.

You can be assured that each cell positive for the reporter construct is also expressing your shRNA or other gene product. Dual expressers can also be useful when delivering secreted gene products, heterodimers, or multivalent vaccines.

Right: an E3 deletion in the backbone (top) allows for ntlacZ and eGFP inserts (middle and bottom).

E3 backbones

Dual-expressing adenovirus in action!

In the image below, a single adenovirus expressing nuclear-targeted B-gal and a secreted protein, TPP-1, was injected into the mouse brain. Serial sections of the cerebellum were stained for b-gal (top) and TPP-1 (below) 10 days post-injection of AdTTP-1/LacZ dual expresser virus. Also present is evidence of retrograde transport of the adenovirus particles from the site of injection in the cerebellum to the brain stem (arrow).

Nuclear targeted bGal demonstrates the distribution of adenovirus transduced cells (top left), whereas the TPP-1 IHC (bottom left) shows the uptake of the secreted TPP-1. Enlarged panels: Note the absence of bGal staining in the choroid plexus (top) but high levels of TPP-1 (bottom).

Haskell RE et al. Gene Ther. 2003 Jan;10(1):34-42. PMID: 12525835

Dual Expressing Virus Constructs

To produce the images below, dual expresser plasmids were designed with an eGFP gene (top panels) and an β-glucuronidase (β-gluc) gene (bottom panels) in the E1 region, and nuclear targeted β-Gal (ntLacZ gene) in the E3 region. Adenovirus encoding these proteins was injected into mouse cerebellum.

Left panel shows transduced cell bodies (GFP fluorescent), whereas right panel shows the infected cell nuclei (β -Gal activity).

Left panel shows distribution of the secreted β-gluc activity emanating from the transduced cell bodies labeled with nuclear targeted β-Gal in the right panel.

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