1 2 3 4 5 6 7 12111098

A549 HeLa

Ad5.CMV5-R1M M

Ad5.CMV5-R1

R1

66

200

97

116

ADENOVATOR™ ADENOVIRAL VECTOR SYSTEMThe Most Powerful Adenoviral Expression System Available

Figure 1: Coomassie blue stained gelof total proteins produced in A549(lanes 1-6) and HeLa (lanes 7-12) cellsinfected with Ad5.CMV5-R1 (lanes 2-6,8-12) and extracted at 48 hours postinfection. Cells were mock infected(lane 1 and 7) or infected at MOIs of 50 (lane 2, 8), 100 (lane 3, 9), 200 (lane 4, 10), 400 (lane 5, 11), and 800 (lane 6, 12). Recombinant R1 production is 25-30% TCP at the highest MOI tested.

Protein Over-Expression inMammalian Cells

Adenoviruses have the advantage of delivering genes

in vitro and in vivo with very high efficiency to a broad host

range and allow for high expression levels of the insert gene.

The adenoviral vector is one of the best systems to use for

gene delivery experiments, protein over-expression in human

cells and gene therapy research.

Advantages

� Highest level of protein expression obtainable by using the unique CMV5 promoter.(1)

� Rapid identification and quantification of co-expressedgenes of interest using GFP or BFP.(2, 3)

� Fast and reliable: Only three easy steps to generate a recombinant Adenovirus — fully compatible with ourAdEasy™ Vector System.(4)

� Comprehensive kit, instruction manual and specialized after-sales technical support.

Increased Protein Expression

The AdenoVator™ Adenoviral Vector System is routinely used

for both protein production and gene transfer experiments.

Among the attractive features of the AdenoVator™ System is

the high gene transfer capacity in a wide range of cell types,

(in vitro and in vivo) and the efficient transgene expression

exhibited. For gene transfer experiments in non-permissive cells,

most Adenoviruses (Ad) make use of the cytomegalovirus (CMV)

immediate-early (IE) promoter-enhancer in the expression cas-

sette, as this promoter is one of the strongest in a wide range

of cell types. Despite this fact, Adenoviruses with CMV-based

expression cassettes rarely produce protein exceeding 1 to 2%

TCP (Total Cellular Protein) after infection of either non-permissive

cells (i.e., A549, HeLa) or 293 cells.(1)

continued on next page

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AdenoVator™ Transfer Vectors allow for the production

of unprecedented levels of recombinant protein in

complementing and non-complementing cell lines.

The main feature of these new vectors is the CMV5

promoter, a combination of enhancer sequences that

increases the transcriptional and translational activities

of the CMV promoter in recombinant Adenoviral

Vectors (AdV). The CMV5 promoter was constructed

by the insertion, downstream of the transcription

start site of the human CMV IE promoter-enhancer,

of the adenovirus tripartite leader (Ad-tpl) with the

adenovirus major late enhancer bracketed by splice

donor and acceptor sites. The Ad-tpl binds translation-

initiating proteins much more efficiently than most

messages. This is a strategy developed by the virus for

efficient translation of the late proteins and is exploited

in these expression vectors. The tpl is composed of

three segments all of which are required for efficient

translation. Although in most cases the presence of

the tpl is thought to facilitate translation, there is

some evidence for its role in improving gene expression

at the level of transcription and message stability.

The pAdenoVator-CMV5 transfer vector significantly

improves the utility of AdV for high-level transgene

expression in mammalian cells and it allows for very

high levels of recombinant protein production in

non-permissive cell lines. This feature greatly simplifies

the production and purification effort, since the

recombinant protein can be produced at levels

approaching 20 to 30% of total cellular protein (TCP)

in the absence of the synthesis of other viral proteins.

The high level of expression achieved with these AdV

in non-permissive cells supports the expression of

transgenes at significant levels in vivo at lower MOIs,

thereby minimizing the toxic side effects.

As shown in Figure 1, the AdenoVator™ System is a

very efficient protein production system allowing

the expression of proteins at near saturation levels in

absence of viral replication in a wide variety of mam-

malian cells. This feature facilitates protein purification

since no other abundant viral proteins are produced.

Moreover, due to the absence of viral replication and

host protein shut-off, the production host can be

maintained in good physiological state for prolonged

periods of time and continue to synthesize the

recombinant protein if secreted in the medium.

Co-expression with GFP and BFP

To improve the utility of the AdenoVator™ Adenoviral

Vector System, coding sequences for the Green and

Blue Fluorescent Protein (GFP and BFP, respectively)

have been included in the optimized pAdenoVator-

CMV5 transfer vectors. These markers of gene

expression have the unique feature of being

detectable in living cells and whole organisms.

Co-expression of GFP or BFP greatly simplifies

the process of recombinant virus identification

and increases the utility of the recombinant Ad.

Generally, recombinant Ads are identified as plaques

on a monolayer of 293 cells that have been overlaid

with agarose. Individual plaques are verified as

being genuinely recombinant by screening methods

that involve plaque elution and amplification followed

by screening (western, PCR, etc.) to verify the presence

of the recombinant protein. By contrast, recombinant

Ads co-expressing GFP or BFP are easily identified by

direct observation with an inverted fluorescent

microscope. Moreover, since GFP or BFP expression

is detectable one day after the transfection of the

recombinant adenovirus DNA in 293 cells, it can be

used to monitor the efficiency of transfection, a

crucial parameter for the successful generation of

recombinant Ads. For in vivo applications GFP or

BFP expression streamlines the identification of

infected cells and so can be used to measure the

effectiveness of the gene transfer protocol.

pAdenoVator-CMV58009 bp

LITR

Kan

RITR

Encapsidation signal

Ori

Right

poly A

Bam HI (1647)

EcoR I (4059)Fse I (4051)

Pme I (1642)

Pac I (1)

Pac I (5076)

arm

Left arm

CMV5 promoter

Figure 2: The Adenovator-CMV5 transfer vectorsignificantly improvesthe utility of AdV for high-level transgene expression in mammalian cells.

pAdenoVator-CMV5 Vector

pAdenoVator-CMV5-IRES-BFP9471 bp

LITR

RITR

Bgl II (1633)

Pme I (5520)

Pac I (6542)

Pac I (1) Encapsidation signal

CMV5

IRE

SB

FP

Kan

Ori

Left arm

Right arm

promoter

poly A

Figure 4: In the AdenoVator™ System the cDNA of interestis first cloned into a transfer vector. The resulting plasmid is

then linearized and co-transformed into E. coli strainBJ5183 together with pAdenoVator ∆E1/E3, the viral DNA

plasmid. This plasmid is E1 and E3 deleted and its E1 functions can be complemented in 293 cells. Upon homol-ogous recombination, clones are selected with kanamycinand screened by restriction enzyme analysis. The recombi-

nant adenoviral construct is then cleaved with Pac I toexpose its ITR (Inverted Terminal Repeats) and transfected

into QBI-293A cells to produce viral particles.

Easy-to-use E. coli-based System

The AdenoVator™ System is a fast and easy alterna-

tive to traditional adenoviral systems and is fully

compatible with the Qbiogene AdEasy™ Vector Kits.

The AdenoVator™ System exploits the robust and

efficient E. coli homologous recombination system.

The construction of a recombinant Ad is a simple

process in which the desired expression cassette is

first assembled into a transfer vector, and subse-

quently transferred into the adenoviral genome by

homologous recombination. Insertion of DNA by

homologous recombination is the most efficient way

of introducing a gene into an AdV because its

genome is too large (36 kb) to be easily manipulated

by conventional molecular biology techniques.

Generation of a Recombinant AdenovirusUsing the AdenoVator™ System

pAdenoVator-CMV5-IRES-GFP9456 bp

LITR

RITR

Bgl II (1633)

Pme I (5505)

Pac I (6527)

Pac I (1) Encapsidation signal

CMV5

IRE

SG

FP

Kan

Ori

Left arm

Right arm

promoter

poly A

Figure 3: In the transfer vectors pAdenoVator-CMV5-IRES-BFP and pAdenoVator-CMV5-IRES-GFPthe reporter genes are encoded as the secondcistron in a dicistronic expression cassette. A geneof interest can be cloned into the first cistron byinsertion into the unique Bgl II site. The vectorcontains two cistrons that are separated by theencephalomyocarditis virus internal ribosomal entry site (IRES).

pAdenoVator-CMV5-IRES-BFP andpAdenoVator-CMV5-IRES-GFP Vectors

2251 Rutherford Road • Carlsbad, CA 92008 USA

phone: 1-760-929-1700 • toll-free: 1-800-424-6101

fax: 1-760-918-9313 • email: technical@qbiogene.com

SDAVVS0J01 © 2000 Qbiogene, Inc.

References1. Massie B, Couture F, Lamoureux L, Mosser DD, Guilbault C, Jolicoeur P, Belanger F, and Langelier Y (1998). Inducible overexpression

of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette. J. Virol. 72: 2289-2296.

2. Mosser DD, Caron AW, Bourget L, Jolicoeur P and Massie B. (1997). Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products. Biotechniques 22: 150-161.

3. Massie B, Mosser DD, Koutroumanis M, Vitte-Mony I, Lamoureux L, Couture F, Paquet L, Guilbault C, Dionne J, Chahla D, Jolicoeur P and Langelier Y. (1998). New adenovirus vectors for protein production and gene transfer. Cytotechnology 28: 53-64.

4. He TC, Zhou S, DaCosta LT, Yu J, Kinzler KW and Vogelstein B. (1998). A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A 95(5): 2509-14.

AdenoVator™ is a trademark of Qbiogene, Inc.

CMV5 promoter is covered under U.S. patent number 5,518,913 exclusively licensed to Qbiogene from the Biotechnology Research Institute,National Research Council of Canada. AdenoVator™ and AdEasy™ products are covered under patent number WO9943843A1 and U.S. patent number 5,922,576 from Johns Hopkins University. Rights to use these products are limited to broader rights or the use of these products for commercial purposes should be directed to Johns Hopkins University School of Medicine Office of Technology Licensing, 2024 E. Monument Street, Suite 2-100, Baltimore, MD 21205. AdEasy™ is a trademark of Johns Hopkins University.

Ordering InformationAdenoVator™ Kits include all of the reagents necessary to generate recombinant Adenoviruses. Choose from our selection of convenient kit formats, from complete starter kits including all reagents and HEK 293 cells, to basic kits and refill supplies to make additional viruses. The highly detailed user manual, with its numerous color pictures and illustrations, is an invaluableresource for beginners as well as those experienced in the field.

AdenoVator™ Adenoviral Vector System Kits

AES2000 .......................AdenoVator™ Basic Kit, including 293 cells, without a transfer vector

AES2000A.....................AdenoVator™ Complete Kit, including 293 cells and pAdenoVator-CMV5 transfer vector

AES2000B .....................AdenoVator™ Complete Kit, including 293 cells and pAdenoVator-CMV5-IRES-GFP transfer vector

AES2000C.....................AdenoVator™ Complete Kit, including 293 cells and pAdenoVator-CMV5-IRES-BFP transfer vector

AES2001 .......................AdenoVator™ Basic Kit, without 293 cells, without a transfer vector

AES2001A.....................AdenoVator™ Kit, without 293 cells, with pAdenoVator-CMV5 transfer vector

AES2001B .....................AdenoVator™ Kit, without 293 cells, with pAdenoVator-CMV5-IRES-GFP transfer vector

AES2001C.....................AdenoVator™ Kit, without 293 cells, with pAdenoVator-CMV5-IRES-BFP transfer vector

Catalog # Description

Components Available Separately

AES2010 .......................pAdenoVator ∆E1-∆E3, the 33.4Kb plasmid containing the ∆E1-∆E3 Adenovirus serotype 5 genome

AES2022 .......................pAdenoVator-CMV5 Transfer VectorAES2023 .......................pAdenoVator-CMV5-IRES-GFP Transfer VectorAES2024 .......................pAdenoVator-CMV5-IRES-BFP Transfer VectorAES1009 .......................Electrocompetent Bacteria BJ5183 + Dh5α, 5 tubes eacAES1005K .....................EC BJ5183 cells, 5 x 80 µLAES1007K .....................EC DH5α, 5 x 40 µLAES0503 .......................QBI-293 HEK cells, 1 mL (1 x 106)AES2015 .......................Refill Kit A: pAdenoVator + BJ5183 EC cellsAES2016 .......................Refill Kit B: pAdenoVator + BJ5183 EC cells + Dh5α EC cells

Catalog # Description