Description of the Method
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.
- 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
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
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 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
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
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.
The basic kit contains the following components:
- QBI-Viral DNA
- QBI-HEK 293A cells
- Transfection reagents (2M CaCl2, TE 0.1X and 2X HBS)
Products Sold Separately
||QBI-HEK 293A cells
||1 mL (1X 106 cells)
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