AdenoQuick 2.0 Kit

Adenovirus vectors with one or multiple transgenes, replication-deficient or oncolytic vectors, WT or modified fiber, E1/E3/E4 deletion, 2 cloning steps in E. coli

  • Most versatile system for adenovirus vector construction
  • Four shuttle plasmids:
    • pAd1127 and derivatives for E1 and pIX genes
    • pAd1128 and derivatives for E2 and late genes
    • pAd1129 and derivatives for E3 and fiber genes
    • pAd1130 and derivatives for E4 genes
  • Ad5 backbone
  • Maximum cargo capacity: 11.2 kb
  • Kit contains: 4 shuttle plasmids, reagents for cosmid construction, adenovirus DNA as positive control for virus rescue
$850.00
Size: 10 reactions

Make your kit

Use our Vector Selection Tool to determine which plasmid is most suitable for your application.

Three steps are needed to rescue a recombinant adenovirus with the AdenoQuick2.0 system:

  1. Shuttle Plasmid Construction:
    Modify one or several of the 4 shuttle plasmids (pAd1127 - pAd1128 - pAd1129 -pAd1130) or their derivatives.
    • pAd1127 contains the Ad5 left ITR, packaging signal, the E1a and E1b regions, and the pIX coding sequence.
    • pAd1128 contains the E2 region (DNA polymerase, terminal protein, DNA-binding protein) and many of the late genes.
    • pAd1129 contains the E3 region and fiber gene.
    • pAd1130 contains the E4 region and right ITR.
  2. Cosmid Construction
    Construct a cosmid containing the entire genome of your recombinant virus:
    • Digest the 4 shuttle plasmids with SfiI, and ligate them to each other. The purification of DNA fragments on agarose gel is not necessary.
    • Package the ligation products into phage lambda, infect E. coli, and select of ampicilin and kanamycin-resistant clones
    • Grow a few colonies, prepare cosmid DNA (= plasmid mini-prep), and confirm their identity by restriction analysis or sequencing
  3. DNA Transfection
    To rescue the  virus, you will need to
    • Linearize the cosmid DNA with PacI or SwaI
    • Transfect the linear cosmid DNA into helper cells (e.g. 293 cells)
    • Harvest plaques, amplify and confirm virus identity

The following is an up-to-date list of the shuttle plasmids pAd1127, pAd1128, pAd1129, and pAd1130, and their derivatives.  Please use our Vector Selection tool to find the plasmid that best suits your needs.

Description Cat # Size

pAd1127

Shuttle plasmid for the construction of adenovirus vectors using the AdenoQuick2.0 system (WT E1 region)
QP-04 20 µg

pAd1127-01

Shuttle plasmid for the construction of replication-deficient adenovirus vectors using the AdenoQuick2.0 system (E1 deletion)
QP-05 20 µg

pAd1127-02

Shuttle plasmid for the construction of replication-deficient, RCA-free adenovirus vectors using the AdenoQuick2.0 system (E1 deletion)
QP-06 20 µg

pAd1127-03

Shuttle plasmid for the construction of conditionally replicative, oncolytic adenovirus vectors (CrAds) using the AdenoQuick2.0 system
QP-07 20 µg

pAd1127-04

Shuttle plasmid for the construction of conditionally replicative, oncolytic adenovirus vectors (CrAds) using the AdenoQuick2.0 system
QP-08 20 µg

pAd1127-05

Shuttle plasmid for the construction of replication-deficient adenovirus vectors using the AdenoQuick2.0 system (E1 deletion with MCS and SV40 polyA signal)
QP-23 20 µg

pAd1127-06

Shuttle plasmid for the construction of replication-deficient, RCA-free adenovirus vectors using the AdenoQuick2.0 system (E1 deletion, complete packaging signal)
QP-24 20 µg

pAd1127-07

Shuttle plasmid for the construction of replication-deficient adenovirus vectors, with a CMV expression cassette in place of the E1 region, oriented towards the right ITR
QP-25 20 µg

pAd1127-08

Shuttle plasmid for the construction of replication-deficient adenovirus vectors, with a CMV expression cassette in place of the E1 region, oriented towards the left ITR
QP-26 20 µg

pAd1127-09

Shuttle plasmid for the construction of replication-deficient adenovirus vectors, with a RSV expression cassette in place of the E1 region, oriented towards the right ITR
QP-27 20 µg

pAd1127-10

Shuttle plasmid for the construction of replication-deficient adenovirus vectors, with a RSV expression cassette in place of the E1 region, oriented towards the left ITR
QP-28 20 µg

pAd1127-11

Shuttle plasmid for the construction of replication-deficient adenovirus vectors with complete packaging signal and CMV expression cassette in place of the E1 region, oriented towards the right ITR
QP-29 20 µg

pAd1127-18

Shuttle plasmid for the construction of adenovirus vectors using the AdenoQuick2.0 system (WT E1 region with 24-bp deletion)
QP-37 20 µg

pAd1128

Shuttle plasmid for the construction of adenovirus vectors using the AdenoQuick2.0 system (WT E2 region and late genes)
QP-09 20 µg

pAd1129

Shuttle plasmid for the construction of adenovirus vectors using the AdenoQuick2.0 system (WT E3 region and fiber)
QP-10 20 µg

pAd1129-01

Shuttle plasmid for the construction of adenovirus vectors using the AdenoQuick2.0 system (1.9 kb E3 deletion and WT Ad5 fiber)
QP-11 20 µg

pAd1129-02

Shuttle plasmid for the construction of adenovirus vectors using the AdenoQuick2.0 system (2.7 kb E3 deletion and WT Ad5 fiber)
QP-12 20 µg

pAd1129-06

Shuttle plasmid for the construction of adenovirus vectors using the AdenoQuick2.0 system (2.7 kb E3 deletion and Ad5/35 fiber)
QP-18 20 µg

pAd1129-10

Shuttle plasmid for the construction of adenovirus vectors, with a CMV expression cassette in place of the E3 region, oriented towards the right ITR
QP-33 20 µg

pAd1129-11

Shuttle plasmid for the construction of adenovirus vectors, with a CMV expression cassette in place of the E3 region, oriented towards the left ITR
QP-34 20 µg

pAd1129-12

Shuttle plasmid for the construction of adenovirus vectors, with a RSV expression cassette in place of the E3 region, oriented towards the right ITR
QP-35 20 µg

pAd1129-13

Shuttle plasmid for the construction of adenovirus vectors, with a RSV expression cassette in place of the E3 region, oriented towards the left ITR
QP-36 20 µg

pAd1130

Shuttle vector for the construction of adenovirus vectors using the AdenoQuick2.0 system (WT E4 region)
QP-13 20 µg

pAd1130-01

Shuttle plasmid for constructing adenovirus vectors with the E4 region under the control of a heterologous promoter
QP-14 20 µg

pAd1130-02

Shuttle plasmid for constructing adenovirus vectors with the E4 region under the control of a heterologous promoter with its own TATA box
QP-15 20 µg

pAd1130-03

Shuttle plasmid for constructing adenovirus vectors with a 2.8 kb deletion in the E4 region
QP-16 20 µg

pAd1130-04

Shuttle plasmid for constructing adenovirus vectors with a 1.2 kb deletion in the E4 region
QP-19 20 µg

Using the AdenoQuick2.0 system to construct your recombinant adenovirus vectors has many advantages:

  1. The cosmid technology that our system uses is particularly well suited for the cloning of the 36 kb-long adenovirus genome in E coli. Because phage lambda packages DNAs ranging from 39 to 54 kb, the cloning method selects bacterial clones containing full-size adenovirus 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:
    • Expression cassettes are linked to a bacterial positive-selection markers (Kanamycin-resistance gene or lambda cos site). Therefore their presence is ensured in the resulting construct. Please note that the Kanamycin-resistance gene is not inserted into the recombinant Ad genome.
    • The SfiI restriction sites used in the procedure generate non-symmetrical cohesive ends suitable for directional cloning.
    • The cosmid construction via packaging into phage lambda generally produces hundreds of clones, with ~ 100% efficiency. The cosmid construction by electroporation is about 70% efficient.
    • Up to 3 µg cosmid DNA per mL bacterial culture can be obtained.
  4. The method is fast:
    • The 4 shuttle plasmids can be modified independently from each other, then assembled together in a single cloning step.
    • Plaques usually appear 7 to 10 days after transfection, sometimes as early as 4 days.
  5. The method is versatile:
    • Each of the 4 shuttle plasmids comes in a growing number of versions.  The result is several hundred possible configurations for your recombinant virus.
    • Two rare-cutting enzymes (PacI and SwaI) are available to linearize the cosmid before transfection into helper cells. Therefore, this method is likely to be useful in a very large number of applications.
  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 cosmid system (~100 %), only a limited number of clones needs to be analyzed at small scale (mini preps).

The AdenoQuick2.0 cloning system is ideal for constructing:

  1. E1-substituted adenovirus vectors, the classical expression adenovirus vector wherein an expression cassette of interest replaces the E1 region.
  2. 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.
  3. E1- and E3-substituted bipartite adenovirus vectors, which contain 2 expression cassettes separated by more than 20 kb DNA: one replacing the E1 region, and the other the E3 region. Examples of applications include:
    • 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
  4. E1/E3/E4-deleted adenovirus vectors, with expression cassettes in the E1 or the E3 regions, or both.
  5. Oncolytic conditionally-replicative adenovirus vectors (CRAd's),  wherein a cell-specific promoter drives the expression of E1a, so that the virus replicates only in that cell type.  Additional transgenes ("therapeutic", killer, reporter genes) can be inserted in other regions of the virus such as E3.
  6. Targeted vectors, wherein the Ad5 fiber has been modified so that the virus can infect other cell types
  7. Vaccine vectors, based on Ad5 or other serotypes such as Ad7, Ad11, Ad26 or Ad35
  • Ad5 backbone
  • Packaging signal:
    • WT (Repeats AI-AVII)
    • Partial (Repeats AI-AV)
  • E1 region:
    • 3.0 kb deletion, corresponding to Ad5 bp 354-3329
    • 3.1 kb deletion, corresponding to Ad5 bp 354-3503
    • 0.1 kb deletion corresponding to Ad5 bp 354-466
  • E3 region:
    • WT
    • dl309 (0.75 kb deletion corresponding to Ad5 bp 30005-30750 substituted with a 642 bp heterologous DNA)
    • 1.9 kb XbaI deletion,corresponding to Ad5 bp 28592-30470
    • 2.7 kb BglII deletion, corresponding to Ad5 bp 28133-30818
  • Fiber gene:
    • WT Ad5
    • hybrid Ad5/35
    • hybrid Ad5/3
  • E4 region:
    • WT
    • 1.2 kb deletion, corresponding to Ad5 bp 35319-35355 and including ORF1-4
    • 2.8 kb deletion, corresponding to Ad5 bp 32830-35649
  • What is a cosmid?

    A cosmid is a large plasmid that was generated by infecting E. coli with bacteriophage lambda.  Cosmids are really great tools to construct recombinant adenoviruses because their minimum sizes (~39 kb) accommodates the 36 kb adenovirus genomes almost perfectly.  When you construct a recombinant adenovirus genome in E. coli using cosmid technology, you are almost sure that every colony carries a full-size genome, and not a smaller plasmid that lost chuncks of the adenovirus genome by DNA recombination.

  • How difficult is it to construct a cosmid?

    Constructing a cosmid is not more difficult, more time-consuming nor more expensive than constructing a plasmid.

    Here are the steps:

    1. Ligate your DNA fragments (2-3 hours at R.T. or overnight at 16 ºC)
    2. Package of the ligation products into lambda phage heads: simply add 2 uL ligation reaction to 6 uL packaging extract (provided in the kit) and incubate at 30 ºC for 90 min.
    3. Infect E. coli with the packaged lambda heads: 30 min at 37 ºC.
    4. Streak on petri dish supplemented with LB + antibiotics - incubate overnight at 37 ºC.
    5. Grow colonies in 2 mL LB/antibiotics (< 12 hrs at 37 ºC)
    6. Purify the cosmid DNA (alkaline lysis method) and analyze by restriction digestion.
  • Which enzyme should I use to linearize my cosmid before transfecting it into helper cells? PacI or SwaI?

    Both PacI and SwaI are rare-cutting restriction enzymes that recognize 8 bp-sequences.  Both of them are flanking both ends of the recombinant adenovirus genome contained in your cosmid.  You should make sure that the enzyme that you will use to linearize your cosmid does not cut inside your transgene.  If neither PacI nor SwaI cuts inside your transgene, there is really no preference.  Because the PacI site is immediately flanking the start of the adenovirus genome, the DNA ends generated by PacI resemble the most 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 4 days after transfecting PacI- or SwaI-digested DNA into 293 cells.

  • What will happen if I do not digest the cosmid DNA with PacI or SwaI prior to the transfection?

    You will most likely not get any virus plaque.  Linearizing the cosmid is important to release the ITRs that serves as origin of replication for the adenovirus DNA polymerase.

  • How do I choose the status of the E3 region: wild-type or deleted?

    In the adenovirus replication cycle, the expression of the E3 region helps the virus evading 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 7.7 kb.

    Therefore, if you are using the most common 3.2 kb E1 deletion, and:

    • if your expression cassette (= promoter + coding sequence + polyA signal) is smaller than 5.0 kb, you can use adenovirus vectors with either wild-type or deleted E3 region. 
    • if your expression cassette is larger than 5.0 kb but smaller than 7.7 kb, you must use E1/E3 deleted vectors.
    • if your expression cassette is larger than 7.7 kb, you must consider E1/E3/E4-deleted adenovirus vectors.

    Notes:

    1. In some applications such as oncolytic adenovirus vectors (CRAds), it might be desirable to retain the entire E3 region,  or 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.
    2. The E3 region can also be used as a location to insert a second transgene, independent from the one inserted in the E1 region.
  • Can I use the AdenoQuick2.0 system if my expression cassette contains a SfiI restriction site?

    Yes, you can!  When you digest your shuttle plasmid with SfiI, you will have to purify two SfiI fragments (instead of one).  As a result, the ligation reaction that you will set up to construct your cosmid will contain 5 DNA fragments (instead of 4).  The cloning efficiency will maybe decrease, but it will work, we have done it.  You might have to verify the integrity of your cosmid with additional restriction digestions, or even sequencing, especially if the additional SfiI site is located inside the coding sequence of your transgene.

  • Can I use the AdenoQuick2.0 system to construct oncolytic adenovirus vectors?

    Our AdenoQuick2.0 system is very versatile.  It allows for mutating practically every region of the Ad5 genome as long as the virus is viable and it is acceptable from safety and bioethics points of view.  For instance you can mutate easily the E1a and E1b genes (e.g. ∆E1ACR2, ∆E1B19k), delete specific E3 genes (e.g. E3b gp19K) and replace them with transgenes so they become activated when the virus replicates, retarget the fiber to specific receptors, mutate the E4 ORFs, etc...

  • The Cosmid Construction Kits are shipped on dry ice. Does it affect the SfiI enzyme it contains?

    Not at all.  The Cosmid Construction Kits -1 and -2 are shipped on dry ice because of the lambda packaging extract.  The SfiI enzyme that is included in the kit will be frozen. We have verified experimentally that a single freeze/thaw cycle will not affect SfiI activity.  However, avoid repeated freeze/thaw.  Once you receive the enzyme, store it at -20 ºC.

  • How do I calculate the maximum cargo capacity of an adenovirus vector?

    You simply sum up the sizes of the deletions present in your vector and add 1.8 kb. For instance, let us suppose that you are considering using an Ad5 backbone characterized by a 3.2 kb E1 deletion (psn 354-3510) and a 2.7 kb BglII E3 deletion (psn 28133-30818).  Your vector will be able to package 3157 bp (= 3510 - 353)   + 2686 bp (= 30818 - 28132) + 1800 bp = 7.6 kb.

    The additional 1.8 kb is the extra DNA that Ad5 capsids can package in addition to their 35,935 bp-long genome (Bett et al, J. Virol. 1993; 67: 5911-21).

  • What is the maximum cargo capacity for the AdenoQuick and AdenoZAP system?

    For the AdenoQuick1.0 system, the current maximum cargo capacity is achieved with E1/E3/E4-deleted pAd362.  The vector allows for inserting 8.9 kb foreign DNA into the E1 region.

    For the AdenoQuick2.0 system, adenovirus vectors can be constructed, in which up to 11.2 kb foreign DNA can be inserted.  It is done by combining the largest E1 deletion (3157 bp in shuttle plasmid pAd1127-02) with the largest E3 deletion and the hybrid Ad5/35 fiber ( 2686 bp + 756 bp in pAd1129-06), and the largest E4 deletion (2815 bp in shuttle plasmid pAd1130-03), plus the extra 1.8 kb that adenovirus capsids can accomodate in addition to the WT 36 kb genome (Bett et al, 1993. J. Virol. 67: 5911-21).

    For the AdenoZAP system, the current maximum cargo capacity of 9.6 kb is obtained with AdenoZAP1.4.

  • Which facilities are required to work with adenovirus vectors?

    The National Institute of Health has designated adenovirus as Level 2 biological agent.  For most applications, working with adenovirus requires therefore a Biosafety Level 2 (BL2) facility.  The NIH guidelines for research involving recombinant DNA molecules stipulate also that experiments which are likely to either enhance the pathogenicity (e.g. insertion of a host oncogene) or to extend the host range (e.g. introduction of novel control elements) of viral vectors under conditions that permit a productive infection should be performed in BL3 facilities.

    A BL2 laboratory should contain:

    • A warning sign on the entrance door limiting the access to authorized persons only.  The sign should identify the agent, list the name and phone number of the lab director or other responsible person, and indicate any special requirement for entering the lab.
    • A Class II biological safety cabinet.  A Class II cabinet is a ventilated cabinet for personnel and product protection having an open front with inward airflow for personnel protection, and a HEPA filtered mass recirculated air flow for product protection.  The face velocity of the inward flow of air through the full-width open front is 75 feet per minute or greater.  
    • At least one tissue culture incubator dedicated to infected cell cultures.  Another separate incubator is desirable for growing uninfected cells.
    • The minimal equipment to handle adenovirus culture without exiting the BL2 lab (such as centrifuges, microscope…).
    • A sink for hand washing
    • A chemical disinfectant kit or at least a gallon of bleach available for spills

    For more information about guidelines, visit this NIH web page.

  • Which precautions should I take while working with adenovirus?

    Work with adenovirus must be performed in a BL2 lab.  There you must:

    • Always wear a lab coat while in the virus lab.  Before exiting the laboratory for non-laboratory areas (e.g. cafeteria, library, administrative offices…), remove your lab coat and leave it in the laboratory.
    • Avoid skin contamination with the virus.  Always wear gloves (one pair OK, two pairs better for added protection).  Once your gloves have been in contact with infectious material, do not touch common appliances such as telephone or doors handles.  Change your gloves frequently.
    • Keep the lab doors closed while work is in progress.
    • Use mechanical pipetting devices.  Do not pipet by mouth.
    • Decontaminate all work surfaces after you finish your work, and immediately after any spill.  Spray a 10% bleach solution, wipe and spray again a 70% ethanol solution.  For large liquid spills, add directly concentrated bleach to the liquid, leave for at least 5 minutes, and wipe.
    • Perform all procedures with infectious particles in the biosafety cabinet to minimize the exposure of personnel to aerosols.  Minimize the creation of aerosols by pipetting virus cultures and suspension very gently.  Use aerosol-resistant tips for pipetting virus suspensions.  Do not conduct work with infectious materials in open vessels on the open bench. 
    • Use needle-locking syringes or disposable syringe-needle units for the injection or aspiration of infectious fluids.  Extreme care should be used to avoid auto-inoculation and aerosol generation.  Needles should not be bent, sheared, replaced in their sheath or guard or removed from the syringe following use.  The needle and syringe should be decontaminated by pipetting in and out concentrated bleach a few times and then promptly placed in a puncture-resistant container.
    • Decontaminate all contaminated liquid or solid wastes before disposal.  Before starting your virus work, pour some bleach into a beaker.  Rinse all materials (tissue culture dishes, pipets, tips…) that came into contact with adenovirus with 10% bleach inside the hood before discarding them in the Biohazard trash and autoclaving. Place all materials to be decontaminated off-site in a durable leakproof container which is closed before removal. If possible, leave the contaminated materials in contact with bleach for a few hours before autoclaving (e.g. after rinsing your pipets with concentrated bleach inside the hood, soak them in a cylinder containing 10% bleach before autoclaving).
    • Do not leave the BL2 laboratory with live viruses, unless they are in a sealed tube.  Cell cultures transduced with adenoviruses should be inactivated either chemically or biochemically before leaving the BL2 facility.
    • Store your adenovirus preparations at –70 °C in closed containers labeled with Biohazard warning signs.
    • Wash your hands when exiting the laboratory.

AdenoQuick2.0 Manual

The AdenoQuick2.0  manual contains all the instructions to construct adenovirus expression vectors using the AdenoQuick2.0 system, including:

  • Shuttle plasmid construction
  • Cosmid construction
  • Virus Rescue

The manual contains also safety guidelines for handling adenovirus vectors, detailed plasmid maps, and troubleshooting guide.