( itself they alsocreate space for up...)Generate Adenovirus Vectors in E. coli by Homologous Recombination,,,with the AdEasy Adenoviral Vector System

Two methods have traditionally been used to generate recombinant adenoviruses. The first involves direct ligation of the gene of interest into the adenoviral genome. The scarcity of unique restriction sites and the prohibitive size of the genome (36 kb) make this method technically challenging. The second and more widely used method involves cloning the gene of interest into a shuttle vector and transferring the gene into the adenovirus genome by means of homologous recombination in vivo.² To isolate and identify recombinant adenovirus by this method, multiple plaque isolations must be performed, which is an extremely laborious and time-consuming process.

The AdEasy system uses a method, developed by T.C. He and colleagues, where homologous recombination between an adenoviral backbone plasmid vector and a shuttle vector carrying the gene of interest is performed in E. coli.³ This method generates recombinant adenoviral plasmid vectors that obviate the need for plaque purification and significantly decrease the time required to generate virus.

Overview of the AdEasy System

In the AdEasy system, the cDNA or expression cassette of interest is cloned into either of two shuttle vectors, pShuttle-CMV^### or pShuttle. pShuttle is used if an alternate promoter, other than CMV, is preferred. Once constructed, the shuttle vector is linearized with Pme I and cotransformed into E. coli strain BJ5183 with pAdEasy-1 viral DNA plasmid. Transformants are selected for kanamycin resistance, and recombinants are subsequently identified by restriction digest. Once a recombinant adenoviral plasmid is identified, it is digested with Pac I to expose the inverted terminal repeats (ITR), then transfected into HEK293
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