Fig. 1. Cloning and production of adenovirus vectors.

Adenovirus vectors containing coding regions for TK2, TK2-DM, dCK-WT, dCK-DM, dCK-A100VTM, HSV1sr39TK (as a reference standard) and Luc2 as a negative control were constructed using Invitrogen’s Gateway^® Cloning System. The open reading frame of each reporter was inserted into an “entry” vector to create the pENTR-PRG plasmids, then transferred to the pAd/CMV/V5/DEST vector, using the LR recombination reaction, to create the seven pAd vectors. The adenoviral plasmids were linearized by PacI restriction enzyme digestion, purified, and transformed into HEK293A cells for vector rescue. After 100% cytopathic effect was observed, lysates were serially passaged on increasing numbers of HEK293A cells with each round of infection until sufficient vector was produced, following cesium chloride buoyant density ultracentrifugation and purification, for in vivo studies. Sites labeled attR1/attR2/attL1/attL2 are the initial recombination regions; attB1/attB2 are the recombination regions after the LR recombination reaction. Cm^R, the chloramphenicol resistance gene; Km^R, the kanamycin resistance gene; Ap^R, the ampicillin resistance gene; ccdB, the coding region for the cytotoxic protein CcdB, used as negative-selection marker in recombined clones; P_CMV, the CMV promoter; TKpA, the thymidine kinase polyadenylation signal; 5’ ITR, the viral 5’ inverted terminal repeats; wt Ad5 (DE3), Ad5 sequences that include a 3’ ITR and packaging signal.