( Efficient gene transfer and high-...)Generate Adenovirus Vectors in E. coli by Homologous Recombination,,,with the AdEasy Adenoviral Vector System

Efficient gene transfer and high-level protein expression within a variety of cells

Tanya Hosfield Lisa Eldridge
Stratagene

Recombinant adenoviruses provide a versatile means of gene delivery and expression. We describe the AdEasy adenoviral vector system,^###,W which simplifies the production of such viruses. Constructing a recombinant adenoviral vector using the AdEasy system is a two-step process in which the desired expression cassette is first subcloned into a shuttle vector, then transferred into the adenoviral genome by homologous recombination in E. coli. This method allows recombinant adenovirus vectors to be selected more quickly and easily than traditional methods.

Recombinant adenoviruses are a versatile tool for gene delivery and expression. Several features of adenovirus biology have made such viruses the vectors of choice for many applications. For example, adenovirus can infect a broad spectrum of cell types, and infection is not dependent on active cell division. The ability to overexpress human proteins in human cell lines allows purification of recombinant proteins with the correct posttranslational processing. Additionally, high titers (amplified titers up to 10¹¹ pfu/ml) and high-level gene expression can be obtained. Consequently, with all these attributes, successful protein production in mammalian cells can be predicted.

The most commonly used adenoviral vector, human adenovirus serotype 5, is rendered replication defective by the deletion of the E1 and E3 genes. The E1 gene is essential for the assembly of infectious virus particles and is complemented in vivo by an adenovirus packaging cell, such as HEK293. The E3 gene encodes proteins involved in evading host immunity and is dispensable. Not only do these deletions prevent the virus from replicating
'"/>

Source: