Adenovirus recombination using BAC

Adenoviruses have large genomes of 36kb, making genetic manipulations by classical cloning strategies extremely difficult and ineffective. SIRION Biotech's in-house Adenovirus cloning system, AdenoONE™ offers a refined solution by benefitting from the large capacity of bacterial artificial chromosomes (BAC). This innovative technology allows for fast and convenient generation of recombinant adenovirus particles.
The system is composed of just two major elements.

  • Component 1: A small shuttle vector (pO6A5) into which the transgene is cloned.
  • Component 2: Electrocompetent BA5-FRT E. coli cells that carry a single BAC vector confering restistance to chloramphenicol. The BAC vector includes the E1/E3 deficient Ad5 genome.
Following transformation of the shuttle vector into the electrocompentent BA5-FRT E.coli cells, Flp recombinase mediated recombination between the shuttle and the BAC vector occurs.

Due to a highly sophisticated selection system, only cells containing recombined BAC vector can grow and form bacterial colonies. As a result almost 100% of screened colonies contain BAC vectors with the correctly recombined transgene. This feature greatly increases the ease of use for the AdenoONE(TM) cloning system as it requires very little ‘hands on’ time from shuttle vector transformation to the isolation of the virus particles. Virus yields and quality are comparable to those obtained with established adenoviral cloning systems.

  1. Cloning of your gene of interest (GOI) or shRNA into the pO6A5 shuttle vector
  2. FLP mediated recombination in E.coliBA5 FRT cells to obtain your desired Ad5 E1/E3 deleted serotype
  3. PAC digestion of of purified recombinant BAC DNA
  4. Virus reconstitution in 293 cells