The AdZ adenovirus cloning system

Biotechniques, 2008

AdZ: A recombinant Adenovirus (Ad) vector with Zero cloning steps

The AdZ system is designed to allow you to clone a gene, shRNA or synthesized sequence directly into a replication deficient recombinant Adenovirus (Ad) vector in a single, simple, recombineering step within E.coli. There is no conventional cloning step; the gene is simply PCR'd then recombineered directionally into the vector, and selectable markers permit the easy identification of positive colonies. Following sequencing, the vector DNA is maxiprepped and transfected into packaging cells in order to grow the recombinant Ad. Through the use of a tet-repression system transgene expression is prevented during vector growth, thus permitting the cloning of toxic gene products. Expression of the transgene in other cell lines is constitutively on. Here you'll find protocols, reagent lists & plasmid maps for use of the AdZ system. 

Transgene Insertion

  • A complete Ad5 vector is carried on a Bacterial Artificial Chromosome (BAC).
  • the DNA insert is transformed directly into cells carrying the AdZ BAC.
  • The insert can be:
    • Synthetic oligonucleotides (e.g. encoding shRNAs)
    • PCR product
    • Synthesized gene
    • Conventional plasmid clone (e.g. from an expression library)
  • Recombineering is performed: the transgene replaces dual selectable marker
  • Positive clones are identified without need for screening.
  • BAC DNA is purified & transfected into cells:
  • AdZ recombinant grows

Additional Features

  • Vectors are optimized for high-throughput applications by making them "self-excising". Incorporation of I-SceI into the BAC vector removes the need to excise vectors prior to transfection.
  • AdZ vectors allow genes to be expressed in their native form or with epitope (strep, V5, eGFP) tags.
  • Insertion of tetracycline operators downstream of the HMCV major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells, allowing the cloning of toxic gene products.

Advantages

  • The AdZ vector system is robust & straightforward.
  • It is suited to occasional use or high-throughput applications.
  • Recombineering provides for directional insertion into the Ad vector.
  • Recombineering allows any modification of the vector backbone to be performed simply and easily