AdenoQuick1-Twin Kit

* E1-deleted adenovirus vectors

* Transgene insertion in the E1 region (max. 7.6 kb cargo capacity)

* Ad5 backbone

* WT E3 or 2.7 kb E3 deletion

* AdenoQuick1.0 cloning system

The AdenoQuick1.Twin kit includes the components necessary to construct at least twenty (20) Ad5-based adenovirus vectors containing sequences of interest in place of the E1 region. Ten viruses can be made with WT E3 region (max. cargo capacity = 5.0 kb). Ten other viruses can be made with a 2.7 kb E3 deletion (max. cargo capacity = 7.6 kb).

The kit includes 3 plasmids:

- pE1.1 is a shuttle vector designed for inserting expression cassettes in place of the E1 region of the Ad5 genome, in combination with the AdenoQuick1.1, AdenoQuick1.2, pAd361 and pAd362 plasmids (construction of first- and second-generation adenovirus vectors). 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 λ cos site, 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. pE1.1 can also be used in combination with vectors pAd329 and pAd330 to construct conditionally replicative adenoviruses (CRAds) containing a heterologous promoter in front of the E1a TATA box.

- AdenoQuick1.1 is a 39.3 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 SfiI sites that allow for directional cloning replace the E1 region. The E3 region is intact. The right ITR is flanked by PacI and SwaI sites. AdenoQuick1.1 is used in combination with the shuttle vectors pE1.1, pE1.11 or pE1.12 to construct replication-deficient adenoviruses containing transgenes in place of the E1 region.

- AdenoQuick1.2 is a 36.6 kb plasmid that contains the sequences encompassing bp 3504-right end (9.8–100 mu) of the Ad5 genome, with a 2.7 kb E3 deletion. 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 SfiI sites that allow for directional cloning replace the E1 region. The right ITR is flanked by PacI and SwaI sites. AdenoQuick1.2 is used in combination with the shuttle vectors pE1.1, pE1.11 or pE1.12 to construct replication-deficient adenoviruses containing transgenes in place of the E1 region.