This kit supplies reagents sufficient to make 5 recombinant adenoviral vectors. The gene to be expressed is inserted into the multiple cloning site of pLEP-CMV, and the plasmid is then cut with a restriction enzyme (PI-Psp I) and ligated to pREP, which contains the remainder of the adenoviral genome. Two versions of the kit are available, one with pREP and the other with pREP-E3. Vectors made with pREP can accommodate inserts up to 5 kb, whereas those made with pREP-E3 can accommodate longer inserts (up to 8 kb). The additional capacity is due to deletion of the adenoviral E3 genes. The E3 genes reduce host immune responses to viral infection, but they are not required for viral propagation. We recommend using pREP whenever possible. The pREP plasmid has been linearized and is ready to be ligated to the pLEP-CMV plasmid. The CMV promoter provides high levels of gene expression in most tissues and cell types. The human growth hormone polyadenylation sequence is used for gene expression. For those who would like to use promoter of their choice along with their gene, we recommend using the pLEP vector (no promoter, no poplyA signal) instead of pLEP-CMV (see K1000 SpeAd^TM kit). The manual that accompanies the kit provides detailed instructions for making adenoviral vectors using the SpeAd^TM method.

Kit includes: pLEP-CMV (20 ug)

pREP7 (or 8), linearized (10 ug)

positive control plasmid to express b-galactosidase (10 ug)

lambda phage packaging extract (5 x 50 ml)

phage dilution buffer (3 ml)

ready-to-use bacterial host (E coli DK-1 cells, 1 ml)

sequencing primers (2, 100 ng each)

SpeAd^TM Manual