Adenovirus Conditional Gene Knockout Vector (Floxed)

The adenovirus conditional gene knockout vector combines VectorBuilder’s highly efficient adenoviral vector system with the Cre-mediated conditional gene knockout system to help you achieve adenovirus-mediated introduction of Cre-mediated inactivation of gene expression into a variety of mammalian cell types. This floxed system comprises LoxP recombination sites flanking a gene of interest to facilitate inactivation of gene expression by Cre-dependent deletion of the coding sequence. In the absence of Cre recombinase, expression of the gene of interest is permitted. When Cre is introduced into cells carrying this vector, the gene of interest is permanently excised.

The adenoviral conditional gene knockout vector is first constructed as a plasmid in E. coli with a LoxP-flanked gene of interest in-between the two inverted terminal repeats (ITRs) during vector construction. It is then transfected into packaging cells along with several helper plasmids where live virus is produced. When the virus is added to target cells, the DNA cargo is delivered into cells where it enters the nucleus and remains as episomal DNA without integration into the host genome. The floxed gene of interest placed in-between the two ITRs during vector construction is introduced into target cells along with the rest of viral genome. Gene expression can then be inactivated in the presence of Cre recombinase upon Cre-mediated deletion of the gene of interest.

By design, adenoviral vectors lack the E1A, E1B and E3 genes (delta E1 + delta E3). The first two are required for the production of live virus (these two genes are engineered into the genome of the packaging cells). As a result, virus produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them). Note this vector alone is not sufficient for achieving recombination between pairs of LoxP sites. Coexpression of Cre is required either via a helper vector or mRNA encoding Cre.

For further information about this vector system and Cre-mediated recombination, please refer to the papers below.

The adenovirus conditional gene knockout vector is designed to achieve Cre-mediated conditional gene deletion in mammalian cells and animals. Expression of the gene of interest is initially under the control of the user-selected promoter and can be permanently silenced by co-expression of Cre recombinase, which will permanently excise the region encoding the gene of interest. This vector is derived from the adenovirus serotype 5 (Ad5). It is optimized for high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression.

Stable gene inactivation: Treatment with Cre recombinase will permanently remove the sequence encoding the gene of interest and prevents its transcription.

Low risk of host genome disruption: Upon transduction into host cells, adenoviral vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

Very high viral titer: After our adenoviral vector is transfected into packaging cells to produce live virus, the virus can be further amplified to very high titer by re-infecting packaging cells. This is unlike lentivirus, MMLV retrovirus, or AAV, which cannot be amplified by re-infection. When adenovirus is obtained through our virus packaging service, titer can reach >10¹¹ infectious units per ml (IFU/ml).

Broad tropism: Cells from commonly used mammalian species such as human, mouse, and rat can be transduced with our vector, but some cell types have proven difficult to transduce (see disadvantages below).

Large cargo space: The upper limit size of the adenovirus genome for efficient virus packaging is ~38.7 kb (from 5' ITR to 3' ITR). After excluding the required backbone components for adenovirus gene expression and Cre-mediated recombination, our vector has about ~7.4 kb of cargo space to accommodate the user's DNA of interest. This is bigger than the ~6.3 kb cargo space in our lentiviral conditional gene expression vector and is sufficient for most applications.

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro. It is particularly suitable for the generation of transgenic animals with Cre-mediated conditional gene knockout.

Safety: The safety of our vector is ensured by the fact that it lacks genes essential for virus production (these genes are engineered into the genome of packaging cells). Virus made from our vector is therefore replication incompetent except when it is used to transduce packaging cells.

Non-integration of vector DNA: The adenoviral genome does not integrate into the genome of transduced cells. Rather, it exists as episomal DNA, which can be lost over time, especially in dividing cells. 

Difficulty transducing certain cell types: While our adenoviral vectors can transduce many different cell types including non-dividing cells, it is inefficient against certain cell types such as endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, and hematopoietic cells.

Strong immunogenicity: Live virus from adenoviral vectors can elicit strong immune response in animals, thus limiting certain in vivo applications.

Technical complexity: The use of adenoviral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming.