Shuttle plasmid for constructing adenovirus vectors with a 2.8 kb deletion in the E4 region

pAd1130-03 is a 7247 bp-long plasmid designed for constructing recombinant adenovirus vectors carrying a 2.8 kb deletion in the E4 region.

It was constructed by deleting the 2.8 kb BsgI-MfeI fragment from plasmid pAd1130.  The deletion encompasses almost the entire E4 region, including the E4 promoter TATA box, ORF1, ORF2, ORF3, ORF4, ORF6, and ORF6/7.  The remaining Ad5 sequences include the right ITR flanked with PacI and SwaI sites, and about 200 bp from the E4 promoter.

pAd1130-03 is used in combination with the AdenoQuick2.0 plasmids pAd1127, pAd1128, pAd1129, or their derivatives, to reconstitute the entire genomes of recombinant adenoviruses in cosmids.  The plasmid contains two SfiI sites that generate non-symmetrical sticky ends suitable for directional cloning, and a 5 kb stuffer sequence made from scrambled phage λ DNA.  This stuffer increases the size of the ligation product of pAd1127, pAd1128, pAd1129, and pAd1130 in order to facilitate DNA packaging into phage λ.

pAd1130-03 can be used with pAd1127-02, pAd1128 and pAd1129-06 to create E1/E3/E4-deleted adenovirus vectors with 11.2 kb DNA cargo capacity.  

Adenovirus vectors carrying the 2.8 kb E4 deletion must be generated and amplified in a cell line expressing the E4 products in trans, such as 293-E4 (Krougliak & Graham, 1995. Hum Gene Ther.6(12):1575-86).

The  sequence of pAd1130-03 was confirmed experimentally.