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Generate Adenovirus Vectors in E. coli by Homologous Recombination,,,with the AdEasy Adenoviral Vector System

combination in E. Coli BJ5183

The AdEasy system is unique to other adenoviral vector systems because homologous recombination between shuttle vector and adenovirus genome is performed in E. coli. The E. coli strain BJ5183 was selected for its recombination capabilities and higher transformation efficiency. To validate production of recombinant adenoviral plasmids in BJ5183, the shuttle vectors described in the previous section were linearized with Pme I, dephosphorylated, and gel purified. Linear shuttle vectors were then electrotransformed into BJ5183 cells, both alone to serve as controls, and cotransformed with the pAdEasy-1 vector to generate recombinants. Miniprep DNA was prepared from picked kanamycin-resistant colonies and analyzed by digestion with Pac I. Positive recombinants were identified by the presence of a large fragment of approximately 30 kb and a smaller fragment of either 3.0 kb or 4.5 kb (depending on whether recombination occurred at the left arm or the origin of replication). Representative Pac I digests of one positively identified recombinant for each of the three shuttle vectors is shown in Figure 3.

Fig.3

High-Titer Virus Production and High-Level Gene Expression

Once recombinant adenoviral plasmid clones were identified, they were digested with Pac I and transfected into HEK293 cells using Stratagenes MBS Mammalian Transfection Kit, with modifications described by Pear and colleagues.4 HEK293 cells are human embryonic kidney cells that have been transformed by sheared Ad5 DNA.5 The cells contain and express the transforming genes of Ad5, including E1, which complements the deleted E1 ge
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