The AdenoQuick™ Cloning System

Overview

The AdenoQuick™ cloning system is an optimized set of plasmids designed to construct:

• First-generation adenovirus vectors (E1-substituted or E3-substituted)
• Second-generation adenovirus vectors ( E1-substituted or E3-substituted, E4 deletion)
• Bipartite adenovirus vectors containing two expression cassettes, one in the E1 region, and the other in the E3 region,
• Oncolytic, conditionally-replicative adenovirus vectors (CRAd's)

Constructing your recombinant adenovirus vector is performed in three easy steps:

Since the construction of large adenovirus plasmids is generally difficult and rebuffing for inexperienced researchers, we have optimized the cloning strategy by using bacterial positive-selection markers (kanamycin-resistance gene and lambda cos site) and a set of specific endonucleases that generate non-symmetrical cohesive ends suitable for directional cloning.

Advantages

Why should you use the AdenoQuick cloning system? Here are 6 reasons:

1. The cosmid technology that our system uses is particularly well suited for the cloning of the 36 kb-long adenovirus genome. Because phage lambda packages DNAs ranging from 39 to 54 kb, the method selects clones containing full-size genomes.
2. No homologous recombination event in E. coli is necessary, thus:
   • There is no danger for an unpredicted recombination in E. coli that would not be detected by restriction analysis and would cause the DNA to be non-infectious.
   • There is no need for the transformation of a recA+ strain (e.g.BJ5183) for the recombination and the subsequent transfer to a recA endA strain for plasmid preparation. Phage lambda infection can be performed directly into recA endA strains such a DH5a, XL-1 blue or Top10.
3. The cosmid construction is very efficient:
   • Each expression cassette is linked to a positive selection marker (Kan or cos site). Therefore its presence is ensured in the resulting construct. Please note that the Kanamycin-resistance gene is not inserted into the recombinant Ad genome
   • The restriction sites used for cloning (SfiI, AlwNI...) generate different sticky ends that allow for directional cloning.
   • The cosmid construction via packaging into phage lambda generally produces hundreds of clones, with almost 100% efficiency. The cosmid construction by electroporation is about 70% efficient.
   • One ml bacterial culture can yield up to 3ug cosmid DNA.
4. The method is fast:
   • Two expression cassettes can be introduced simultaneously into the viral genome.
   • Plaques usually appear 7 to 10 days after transfection, sometimes as early as 4 days.
5. The method is versatile:
   • Six different enzymes are available to excise the expression cassettes from the shuttle vectors.
   • Two rare-cutting enzymes (PacI and SwaI) are available to linearize the cosmid before transfection into helper cells (such as 293...). Therefore, this method is likely to be useful in a very large number of applications.
   • The reconstitution of the genome of the recombinant virus in a cosmid can be performed either via packaging into phage lambda or by electroporation.
6. Compared to other techniques that also reconstitute the entire sequence of the recombinant virus in plasmid, AdenoQuick requires less “hands-on” time: given the very high percentage of correct clones obtained using the lambda system (~100 %), only a limited number of clones needs to be analyzed at small scale (minipreps). Experienced users can directly set up a midi-prep or maxi-prep in order to obtain transfection-grade DNA.

Applications

The AdenoQuick™ cloning system is ideal for constructing:

• E1-substituted adenovirus vectors (the classical "First-Generation" adenovirus vector).
• E3-substituted adenovirus vectors. This type of vector can be used to avoid the emergence of replication-competent adenoviruses (RCA) during virus amplification in 293 cells. The expression cassette of interest is inserted into the E3 region, while the E1 region is kept empty. If necessary the size of the vector is brought to about 36 kb using stuffer DNA. If the viral vector recombines with the WT E1 region inserted into the 293 cell chromosome, a viral genome will be generated, which will be too long to be packaged, and will not generate viral particles. Please note that there are some restrictions about the nature of the expression cassette that can be inserted into the E3 region.
• E1- and E3-substituted bipartite adenovirus vectors, e.g.:
  • Vectors containing your gene of interest in the E1 region, and a reporter gene in the E3 region, or vice-versa
  • Vectors containing inducible expression systems
  • Vectors expressing a regulator and a regulated gene
  • Vectors expressing two genes involved in the same pathway
• E1/E3/E4-deleted adenovirus vectors, with expression cassettes in the E1 or the E3 regions, or both (so-called "second generation" adenovirus vectors)
• Oncolytic conditionally-replicative adenovirus vectors (CrAd's).

Features

• Ad5 backbone
• E1 deletion (3.1 kb, correcponding to bp 354-3,503 in the Ad5 genome, or 0.1 kb, corresponding to bp 354-466 in the Ad5 genome)
• WT or deleted E3 region (2.7 kb, corresponding to bp 28,133-30,818 in the Ad5 genome)
• WT or deleted E4 region (1.2 kb, corresponding to bp 35,319-35,355 in the Ad5 genome, including ORF1-4)
• The table below summarizes the cargo capacities of our vectors

Available Systems

The AdenoQuick™ cloning system includes 9 large adenovirus plasmids and 3 shuttle vectors. The table below offers a synopsis of the different combinations of shuttle vector/adenovirus plasmid, applications, maximum cargo capacity, and selected features (E1, E3, and E4 region status).

* Maximum Cargo is calculated taking into account the fact that Ad5 can package about 1.8 kb heterologous DNA without compromising virus growth.

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