This product pack contains the plasmids required for Adenovirus construction and a HEK-293 cell line to allow virus recovery. The plasmids allow genes to be efficiently inserted into the E1 region using either homology based or restriction digest based approaches.
This product contains the plasmids and cell line required for the construction of Adenoviral (Ad5) viral vectors for expression of genes in a wide range of cell lines.
Method of Use:
This kit works by providing an Ad5 E1/E3 deleted genome in plasmid OG268 that contains restriction sites in the E1 region. This allows expression cassettes to be cloned in using either restriction site based methods or homology based methods. The plasmid contains a small multiple cloning site consisting of AsiSI ClaI and PacI. These sites can be found in this order in the majority of our catatalogue plasmids, facilitating easier virus contruction.
The kit also contains a shuttle plasmid that contains arms of homology to the regions flanking in the restrictions sites in OG268, the use of this plasmids can signficantly improve the recombination efficiency for virus construction.
The kit also contains a vial of HEK-293 cells to allow virus recovery because the HEK293 line expresses both Adenovirus E1A and E1A genes.
The wildtype virus genome is 35938 base pairs but in this vector the genome has been shortened by the deletions to make it 30501 base pairs. This allows for the insertion of up to 5.5kb of exogenous DNA. It has also previously been shown that serotype 5 adenovirus genomes can accomodate insertions up to 105% total genome length compared to wild type. For this reason it should be possible to insert an additional 1.7kb resulting in a total insertion size of 7.2Kb. We have previously packaged viruses up to 105% of the virus genome with good stability but some insertions may be unstable at this length depending on sequence. These numbers are therefore provided as guidelines only.
Recovery and Growth:
Virus recovery requires two steps. Firstly the plasmid must be cleaved with the SwaI restriction enzyme to linearise the virus genome. Then the linear genome must transfected into cells to recover the virus. This can be performed in HEK-293 cells or any derivative of HEK-293 cells such as HEK-293As and HEK-293T cells. These lines contain and express the adenovirus E1A protein that trans-compliments the deletion in the E1A region in this plasmid. A typically recovery transfection would use approximately 5ug of DNA. It is not necessary to remove the plasmid backbone after linearising the DNA a standard DNA clean up will be sufficient rather than gel extraction. Virus colonies should appear approximately 10-15 days after transfection into 293 cells however clones can sometimes appear at earlier time points. Cell should be cultured in 2% fetal calf serum (FCS) to prevent over growth during this time.
The purchase of this kit provides the user with a research license only, in either an academic or research institution. These licenses do not allow the production of Adenoviral vectors for third parties or for profit. If you wish to use these vectors in this context please contact us.