Why do I have to use PIR1 cells for the transformation of the pO6A5 vector from the ibiClone Adenovirus Cloning Kit?

Using PIR1 cells is mandatory, because pO6A5 vectors possess the R6K origin of replication and this allows plasmid propagation only in these cells.

What makes adenoviral vectors such suitable tools for gene expression experiments?

Due to the broad host tropism of adenoviruses, they are ideal vectors for transferring genes into the majority of mammalian cells. Adenoviruses are episomal vectors that do not integrate into the host genome. They allow for the transient, high-level expression of recombinant proteins, and they also mediate very efficient gene knockdowns. The ability of the adenoviral vectors to be purified to high titers (up to 1013 virus particles per ml) makes them ideal tools for in vivo applications. Also, as adenoviruses possess the important attribute of stability in the bloodstream, they can be employed for gene delivery, following intravenous administration.

How can I prevent clogging in the column that is used in the ibiPure Adenovirus Purification Kit?

The reason for the clogging of the column is that there is too much cell debris in the viral lysate. Perform the following steps to prevent clogging:

  • Increase the centrifugation time.
  • Pipet the lysate that is loaded onto the column up and down, and then repeat the centrifugation step.
  • Try adding the 10x Loading Buffer with more care.
  • Do not freeze/thaw the viral lysate and the cell pellet after the final centrifugation step.

How do I store purified adenovirus samples?

After the purification of the adenovirus, using the ibiPure Adenovirus Purification Kit, store the purified adenovirus samples at -80 °C.

How does the ibiBoost Adeno Adenovirus Transduction Enhancer work?

ibiBoost Adeno is based on an adenovirus binding peptide. When added to adenovirus particles, ibiBoost changes the adenovirus surface in a way that the uptake of adenovirus is improved for all permissive cell types, and it allows transduction of cell types previously not accessible to adenovirus transduction. Unlike chemical transduction enhancers, ibiBoost has no toxic effects. The use of ibiBoost is especially recommended if high ”Multiplicities of Infection“ (MOI), exceeding 500, have to be applied to a particular cell type.