Fig. 1
From: Efficient Generation of Recombinant Adenoviral Vectors by Cre-lox Recombination In Vitro
Description of the adenovirus construction system by Cre-loxP recombination in vitro. (A) Schematic outline of the Cre-loxP system. The shuttle plasmid contains the adenoviral 5′-ITR and packaging signal from 0 to 1 mu, the gene of interest, and a single loxP sequence. The adenoviral cosmid has a single loxP sequence and 9.2–100 mu of adenovirus genome with a deletion in the E3 region. First, the gene of interest is cloned into a shuttle plasmid, then the resultant shuttle plasmid is linearized by a restriction enzyme like NheI and recombined with ClaI-digested adenoviral cosmid in vitro. Cre recombinase can produce the full-length recombinant adenoviral vector in vitro by an exchange of regions distal to the loxP site linearized in these two molecules. The recombined DNA is transfected into packaging cell lines as 293 cells, and homogeneous recombinant adenovirus can be produced within 7–9 days without plaque purification. (B) Cre-mediated recombination of pAdCMVlacZloxP and cS360loxP in vitro. Equal numbers of molecules of shuttle plasmid and adenoviral cosmid were treated with or without Cre in vitro, and 500 ng of DNA was fractionated on a 0.4% agarose gel, transferred on a nylon membrane, and analyzed with a 32P-labeled lacZ probe. The upper arrow indicates the full-length of adenoviral DNA arising from recombination. The bottom arrow indicates pAdCMVlacZloxP plasmids. Lane 1: DNA not incubated with Cre; lane 2: Cre-treated DNA.