The AdenoZap™ Cloning System
Overview
The AdenoZap™ Cloning System is based on the in vitro ligation of two DNA molecules. The first molecule which we refer to as the "left arm" contains the adenovirus left ITR, packaging signal and your expression cassette. The second DNA molecule, which we refer to as the "right arm", contains most of the adenovirus genome (from bp 3504 to the right end, with or without a 2.7 kb E3 deletion).
The right arm is provided in the kit. It originates from a bacterial cosmid rather than from virions, thereby eliminating the danger for a possible viral contamination. It will generate virus only after being ligated to the left arm that contains your expression cassette and the packaging signal.
We have designed the AdenoZap™ cloning system in such a way that the ligation of both left and right arms is directional. Because of the non-symmetrical nature of the sticky ends generated by excising the left arm from the shuttle vector, the ligation of the left and right arms should generate only one type of viral DNA molecule. Consequently, a homogeneous virus population should be generated upon transfection of the helper cells. In addition, such ligation is performed to almost completion in less than an hour using an ordinary T4 DNA ligase.
The ligation product is directly transfected into a helper cell line such as 293 cells. Viral plaques usually appear 7 days after transfection, sometimes as early as 3 days.
Compared to other methods currently available, the AdenoZap™ System has the big advantage of requiring only one cloning step in E. coli. The only cloning you have to perform in E. coli is inserting your expression cassette into a small shuttle vector. Thus no large plasmid (or cosmid) construction and purification is necessary. This will save you a lot of time and money.
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Choosing the AdenoZap™ Cloning Kit Suitable for your application.
The AdenoZap™ Cloning System exists in different versions and each version can be used in different ways. The following table will help you decide which one is the most appropriate to your application.
Table 1 : Overview of the Various AdenoZAP Cloning Systems.
Kit |
Shuttle Vector |
Enzymes available for Left Arm Excision |
Right Arm |
E3 Region Status |
Maximum Cargo Capacity |
|
Left End |
Right End |
|||||
AdenoZAP1.1 |
Pac I, SwaI* |
Sfi I, BstAPI, DraIII, AlwNI, Pfl MI |
WT E3 |
5.2 kb |
||
AdenoZAP1.2 |
Pac I, SwaI* |
Sfi I, BstAPI, DraIII, AlwNI, Pfl MI |
Δ E3 |
7.9 kb |
||
AdenoZAP2.1
|
Pac I, SwaI |
I-SceI |
WT E3 |
5.2 kb |
||
AdenoZAP2.2
|
Pac I, SwaI |
I-SceI |
Δ E3 |
7.9 kb |
||
* SwaI is not available for AdenoZAP1.1 and 1.2, lots 0101.
AdenoZAP1.X or AdenoZAP2.X?
Both the AdenoZap1.X and AdenoZap2.X cloning systems rely on endonucleases that generate non-symmetrical sticky ends with which directional ligation can be performed. The only difference between both systems is the nature of the enzymes available to excise the left arm from the shuttle vector pZAP. In pZAP1.1, the left arm can be excised using PacI or SwaI on the left side, and either SfiI, BstAPI, DraIII, AlwNI or PflMI on the right side. PacI and SfiI are 8-base cutters while BstAPi, DraIII, AlwNI and PflMI are 6-base cutters. In pZAP2.1, the left arm can be excised using PacI or SwaI on the left side and I-SceI on the right side. I-SceI is an intron-encoded endonuclease that recognizes an 18-bp-long sequence within which minimal sequence degeneracy is tolerated.
PacI or SwaI?
The AdenoZap System offers the choice between PacI and SwaI for digesting pZAP at the border of the left arm. Both enzymes are 8-base cutters, thus they should be present at equal frequencies in DNA. The only difference is the position of the restriction sites relative to the start of the adenovirus left ITR. The adenoviruses that will be generated from either PacI or SwaI-linearized DNAs will be identical: their genome will start with the correct nucleotide. Indeed, the replication of adenovirus DNA is a protein-primed mechanism where an intermediate: the pre-terminal protein covalently linked to the first three nucleotides CAT, is systhesized opposite to positions 4-6 before jumping back to position 1 of the template to start elongation.
The DNA ends generated by PacI resemble most of the ends obtained from deproteinised virion DNA and might therefore be more efficient in promoting virus replication.
In practice however, no difference in the time needed to recover the virus after DNA transfection into 293 cells is observed between both settings. Virus plaques can appear as early as 3 days after trnsfecting PacI- or SwaI-digested DNA into 293 cells.
WT E3 or ΔE3?
In the virus replication cycle, the expression of the E3 region helps the virus to evade the host immune system. This region is not essential for virus replication in vitro and therefore can be deleted in order to construct adenoviruses containing longer transgenes, up to 8.2 kb. However, in some applications, it might be desirable to retain or even increase the expression of some E3 products: for instance, the adenovirus “death protein” E3-11.6K, which facilitates the release of viral particles from infected cells, or gp19K, whose constitutive expression reduces the host cytotoxic T cell response against the vector and increases the persistence of transgene expression on its own but possibly not in the context of constitutive expression of the entire E3 region.
The E3 region can be used to insert a second transgene, independent from the one inserted in the E1 region.
TopReferences
Follow this link to a selection of publications that utilize O.D.260 technologies.