Figure 2.

Generating the Ad-Array

(A) >300 P. yoelii pre-erythrocytic genes were amplified using P. yoelii genomic DNA and gene-specific primers. PCR products were electrophoresed on 1% agarose gels, showing a subset of 24 genes. The control is a pair of oligonucleotide primers used to amplify the E1 region of Ad5 DNA. (B) Parallel generation of two Ad-array vectors in multi-well plates. The schematic indicates two Ad-array vectors, AdgPyHep17 and AdgCMVp65. The inverted terminal repeats (ITRs), CMV promoter, SV40 poly(A), and the 25-bp-long attB recombinase sequences that flank the transgene (B1 and B2) are indicated. Two plasmids, encoding PyHep17 and CMVp65, were AdFlex linearized with Pac I and transfected into 293 cells in 60-mm, 6-well, 12-well, 24-well, 48-well, and 96-well plates. Following two passages in 293 cells in the same plate size, CPE was observed in all wells. Viral DNA was obtained, and PCR analysis was performed using primers that flank the expression cassette as indicated by the arrows in the schematic. The products of the PCR reaction were loaded into a 1% agarose gel and electrophoresed. Arrows next to AdgPyHep17 and AdgCMVp65 indicate the expected size for the PCR products.