( cells where the E1 gene (necessary for ...)Generate Adenovirus Vectors in E. coli by Homologous Recombination,,,with the AdEasy Adenoviral Vector System

Fig.1

Vector Description

The pAdEasy-1 vector contains most of the human adenovirus serotype 5 (Ad5) genome and is deleted for the genes E1 and E3 (Figure 2). The expression cassette from the shuttle vector is inserted into the original E1 region of the adenovirus genome by homologous recombination. The pAdEasy-1 vector carries the ampicillin resistance gene, which is lost after recombination with a shuttle vector.

Fig.2

The pShuttle-CMV vector contains a multiple cloning site between the CMV promoter and the SV40 polyadenylation signal. The pShuttle vector contains only a multiple cloning site in which an entire expression cassette can be inserted. The regions indicated as arms are the stretches of sequence homology with pAdEasy-1 vector where homologous recombination occurs. The RITR and LITR regions are short inverted terminal repeats (Right and Left) and have a role in replication of the viral DNA.² Both of these shuttle vectors contain the kanamycin resistance gene.

Three test shuttle vectors, pShuttle-CMV-GFP, pShuttle-CMV-LacZ, and pShuttle-CG were prepared to validate the AdEasy system described below. The pShuttle-CMV-GFP vector was prepared by cloning the sequence for green fluorescent protein (GFP) between the Bgl II and Xho I sites into pShuttle-CMV vector. The pShuttle-CMV-LacZ vector was prepared by cloning the sequence for E. coli LacZ between the Xho I and Hind III sites in pShuttle-CMV vector. The pShuttle-CG vector was prepared by inserting an entire expression cassette, which included the CMV promoter, and the green fluorescent protein between the Xho I and Sal I sites in the pShuttle vector.

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