AdenoQuick13.1 Kit

* E1/E3-deleted adenovirus vectors

* Dicistronic (a.k.a. bipartite) vectors

* Transgene insertion in the E1 region and E3 region

* max. 7.5 kb cargo capacity

* Ad5 backbone

* 2.7 kb E3 deletion

* AdenoQuick1.0 cloning system

The AdenoQuick13.1 kit includes the components necessary to construct at least ten (10) Ad5-based bipartite adenovirus vectors containing two transgenes, one in place of the E1 region, and the other in place of the E3 region. The maximum combined cargo capacity is 7.5 kb.

The kit includes 3 plasmids:

- pE1.2 is a shuttle vector designed for inserting expression cassettes in place of the E1 region of the Ad5 genome, in combination with the pE3.1 and AdenoQuick13.1 or pAd363 plasmids. It contains the first 353 base pairs (map unit 0-1) from the Ad5 genome (including the left ITR and packaging signal), preceded by PacI and SwaI sites, and followed by a multiple cloning site. Expression cassettes inserted into this site should contain a promoter, coding sequence and a polyA signal. The sequences encompassing the kanamycin-resistance gene, the adenovirus 0-1 map units and the multiple cloning site are flanked by two sets of restriction sites AlwNI, BstAPI, DraIII and PflMI which generate incompatible and non-symmetrical sticky ends. pE1.2 can also be used in combination with plasmids pAd328 and pE3.1 to construct "armed" conditionally replicative adenoviruses (CrAds) containing a heterologous promoter in front of the E1a TATA box, and a transgene in place of the E3 region.

- pE3.1 is a shuttle vector designed for inserting expression cassettes in place of the E3 region of the Ad5 genome, in combination with the pE1.2 and AdenoQuick13.1 or pAd363 plasmids. It contains a multiple cloning site (MCS) adjacent to a 178 bp-long λ cos site. Expression cassettes inserted into the MCS should contain a promoter, coding sequence and a polyA signal. The sequences encompassing the cos site and the MCS are flanked by two sets of restriction sites AlwNI, BstAPI, DraIII and PflMI which generate incompatible and non-symmetrical sticky ends. pE3.1 can also be used in combination with plasmids pAd328 and pE1.2 to construct "armed" conditionally replicative adenoviruses (CRAds) containing a heterologous promoter in front of the E1a TATA box, and a transgene in place of the E3 region.

- AdenoQuick13.1 is a 36.7 kb plasmid that contains the sequences encompassing bp 3504-right end (9.8–100 mu) of the Ad5 genome. The two SfiI sites naturally present in WT Ad5 DNA were mutated by substituting A for G and C at positions 16291 and 16294 in the Ad5 genome, and C and G for respectively G and C at positions 23001 and 23004 in the Ad5 genome, introducing silent mutations in the adenovirus pVII and DNA-binding protein coding sequences. Two pairs of SfiI sites that allow for directional cloning replace the E1 and E3 regions. The size of the E3 region deletion is 2.7 kb. The right ITR is flanked by PacI and SwaI sites. AdenoQuick13.1 is used in combination with the shuttle vectors pE1.2 and pE3.1 to construct bipartite replication-deficient adenoviruses containing transgenes in place of the E1 and E3 regions. The maximum combined transgene capacity is 7.5 kb.