Adeno-Quest™ Technology

Description of the Method
This system is based on in vivo homologous recombination in human QBI-HEK 293A cells. The principle of the system is simple: the gene of interest, or the complete expression cassette (including promoter, gene of interest and poly A signal), is sub-cloned into a transfer vector, which is introduced, together with linear viral DNA, into human QBI-HEK 293A cells. The subsequent in vivo homologous recombination will yield recombinant viral particles. After isolation, pure recombinants from rounds of plaque purification will be transduced into QBI-HEK 293A cells for amplification.

Advantages

• Investigators who are more confident in homologous recombination in human 293 cells than homologous recombination in bacterial cells or transposition can use this system instead
• Proven method that has given full satisfaction to many researchers over a number of years

Viral DNA and Cell Lines Used

QBI-Viral DNA
The adenoviral DNA used in this kit is derived from the serotype 5, subtype dl327. It is produced from a recombinant adenovirus that contains the bacterial lacZ gene under the control of the human CMV promoter and from which the E1 and E3 regions of Ad5 have been deleted (Ad5.CMV-LacZE1/E3). The viral DNA is Cla I cut in order to remove the left ITR, the packaging signal for the virus, and the lacZ gene, rendering the viral DNA unable to replicate in cells. Enough DNA is provided to produce 5 recombinant viruses.

QBI-Transf
QBI-Transf consists of uncut viral DNA as described above (QBI-Viral DNA). After transfection into QBI-HEK 293A cells, the viral DNA is replicated and virus is produced. The QBI-Transf DNA is used to monitor the efficiency of the calcium phosphate transfection into QBI-HEK 293A cells.

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.

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 replication and amplification.

Kit Information
The basic kit contains the following components:

• QBI-Viral DNA
• QBI-Transf
• QBI-Infect
• QBI-HEK 293A cells
• Transfection reagents (2M CaCl₂, TE 0.1X and 2X HBS)

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