AdenoVator™Adenoviral Vector System

Description of the Method
Qbiogene Molecular Biology's AdenoVator™ Adenoviral Expression System is based on homologous recombination in E. coli cells (BJ5183) between a transfer vector and a plasmid containing the E1/E3 deleted Ad5 genome (pAdenoVator E1/E3). The gene of interest is cloned into the transfer vector, which is subsequently co-transformed with the Ad5-backbone containing plasmid into BJ5183 cells where the homologous recombination will take place. Upon picking of positive recombinants, the recombined vector is extracted and then introduced into DH5 EC (Electrocompetent) cells for amplification. The next step consists of extracting the recombinant plasmid from the DH5 cells, linearizing the DNA, and transferring it into QBI-HEK 293A cells to ensure the production and amplification of viral particles.

Advantages

• Gene transfer accomplished using the highly efficient homologous recombination apparatus of E. coli cells
• No plaque purification required
• Large choice of transfer vectors
• Can be used in combination with the Q-mate™ Inducible Expression System
• Highest level of protein expression available by using the CMV5 promoter (up to 35% of total cellular protein)
• Rapid identification and quantification of co-expressed genes using GFP or BFP
• 100% compatible with the AdEasy™ adenoviral vector system
• Fully compatible with the BMAdE1™ RCA free cell line

Q-mate™ controllable Transfer vectors
Qbiogene now also offers several transfer vectors designed for controllable gene expression using the Q-mate™ Inducible Expression System.

These vectors contain the operating sequences for the CymR repressor (CuO) linked to either the CMV or the CMV5 promoter, which is 5 to 10 times stronger than the CMV promoter. pAdenoVator-CMV5(CuO)-IRES-GFP carries the IRES sequence and the GFP coding region which allow the co-expression of the protein of interest and the GFP.

See Q-Mate™ slide presentation

off	on
Generation of recombinant Adenoviruses expressing toxic proteins using the Q-mate™ Inducible Expression System. QBI-HEK 293CymR cells infected with Ad5.CMV5(CuO)-VSVG in the absence (OFF) or presence (ON) of cumate.

Generation of a recombinant Adenovirus (rAd) expressing a toxic protein. Tight control of expression of a toxic protein in a transfection assay. QBI-HEK 293-CymR cells, stably expressing the repressor molecule CymR, were transfected with pAdCMV5(CuO)-VSVG in the presence (ON) or absence (OFF) of cumate. Cell extracts were prepared 48h post transfection for Western analysis.

CMV5: The CMV5 promoter was constructed by the insertion, downstream of the transcription start site of the human CMV IE (immediate early) promoter-enhancer, of the tripartite leader (tpl) and the splicing sites present in the human Adenovirus serotype 5 Major Late Promoter (MLP). The tpl binds translation-initiating proteins much more efficiently than most messages, resulting in a significant increase in protein expression levels compared to a standard CMV promoter.

IRES (Internal Ribosome Entry Segment): sequence supporting translation from the second cistron in a dicistronic message.

Cell Lines and Viruses Used

BJ5183 EC
BJ5183 EC are electrocompetent (EC) Escherichia coli cells used in the AdenoVator™ and AdEasy™ systems for homologous recombination. This strain is not suited for amplification of recombinant plasmid.
Genotype: endA sbcBC recBC galK met thi-1 bioT hsdR (Str^r).

DH5 EC
DH5 EC are electrocompetent Escherichia coli cells used in the AdenoVator™ and AdEasy™ kits for amplification of recombinant plasmid. This strain is not suited for homologous recombination.
Genotype: F- deoR recA1 endA1 hsdR17(rk^-, mk ) supE44 ^- thi-1 gyrA96 relA1.

QBI-HEK 293A Cells
The QBI-HEK 293A cell line is a permanent line of primary human embryonic kidney (HEK) cells transformed by sheared human Ad5 DNA. These cells contain the E1A and E1B Ad5 viral genes, which complement the deletion of this essential region in the recombinant adenovirus. We have selected a superior sub-clone of HEK 293 that adheres strongly to plastic dishes and performs extremely well in plaque assay and transfection experiments. All our adenoviral vector systems use QBI-HEK 293A cells for viral replication and amplification.

QBI-Infect Viral Particles
The QBI-Infect is a first generation adenovirus expressing the bacterial LacZ gene under the control of the immediate/early CMV promoter (Ad5.CMVLacZE1/E3). This virus can be used to perfect adenovirus culture techniques before producing recombinant virus. People inexperienced with virology techniques can use QBI-Infect to prepare control plaques. Once amplified, QBI-Infect Adenovirus can be used to test the infectivity of cell lines or tissues other than QBI-HEK 293A cells or commonly used eukaryotic cells. The success of DNA transfer can be easily monitored with a substrate for -galactosidase. The QBI-Infect Viral Particles are included in all of our adenoviral vector systems.

Kit Information
The basic kit contains the following components:

• BJ5183 cells
• DH5 cells
• pAdenoVator E1/E3
• QBI-Infect
• Transfection reagents (2M CaCl₂, TE 0.1X and 2X HBS)

Products Sold Separately

Cat #	Description	Quantity
AES2010	pAdenoVator DE1/E3, the 33.4 kb plasmid containing the DE1/E3 Adenovirus serotype 5 genome	2.5 µg
AES2022	pAdenoVator-CMV5 transfer vector	25 µg
AES2023	pAdenoVator-CMV5-IRES-GFP transfer vector	25 µg
AES2024	pAdenoVator-CMV5-IRES-BFP transfer vector	25 µg
AES2025	pAdenoVator-Promoterless transfer vector	25 µg
AES2026	pAdenoVator-Promoterless-Intron transfer vector	25 µg
AES2027	pAdenoVator-hCMV transfer vector	25 µg
AES2028	pAdenoVator-hCMV-Intron transfer vector	25 µg
AES2029	pAdenoVator-RSV transfer vector	25 µg
AES2030	pAdenoVator-SV40 transfer vector	25 µg
AES2033	pAdenoVator-CMV5-(N)His-1	25 µg
AES2034	pAdenoVator-CMV5-(N)His-2	25 µg
AES2035	pAdenoVator-CMV5-(N)His-3	25 µg
AES2036	pAdenoVator-CMV5-(C)His-1	25 µg
AES2037	pAdenoVator-CMV5-(C)His-2	25 µg
AES2038	pAdenoVator-CMV5-(C)His-3	25 µg
AES2039	pAdenoVator-CMV(CuO)	25 µg
AES2040	pAdenoVator-CMV5(CuO)	25 µg
AES2041	pAdenoVator-CMV5(CuO)-IRES-GFP	25 µg
AES1009	BJ5183 EC DH5a EC	5 tubes each
AES1005K	BJ5183 EC	5 X 80 µL
AES1007K	DH5a EC	5 X 40 µL
AES0503	QBI-HEK 293A cells	1 mL (1X 106 cells)
AES2044	QBI-HEK 293CymR cells	1 mL (1X 106 cells)
AES2046	Cumate	(200 mg/mL)
		1 mL
AES2047	Cumate	(200 mg/mL)
		10 mL
AES2015	Refill kit: pAdenoVator E1/E3 BJ5183 EC	Kit
AES2016	Refill kit: pAdenoVator E1/E3 BJ5183 EC DH5a EC	Kit

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