Building Virus Vectors from scratch with BAC
Viral vectors can have large genomes (i.e. AV 36kb) making genetic manipulations by classical cloning strategies extremely difficult and ineffective. SIRION Biotech’s in-house virus cloning technology offers a refined solution by benefiting from the large capacity of bacterial artificial chromosomes (BAC).
This innovative technology allows for fast and convenient generation of recombinant viral particles.
The system is composed of just two major elements.
- Component 1: A small shuttle vector (pO6A5) into which the desired transgene is cloned.
- Component 2: Electrocompetent BA5-FRT E. coli cells that carry a single BAC vector confering resistance to chloramphenicol. The BAC vector includes the replication deficient viral genome.
Following transformation of the shuttle vector into the electrocompetent 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 cloning system as it requires very little ‘hands on’ time from shuttle vector transformation to the isolation of the virus particles. When applied to adenoviral vectors the virus yields and quality are comparable to those obtained with established adenoviral cloning systems.
Example of the BAC technology using E1/E3 deficient Ad5 genome.
- Cloning of your gene of interest (GOI) or shRNA into the pO6A5 shuttle vector
- FLP mediated recombination in E.coliBA5 FRT cells to obtain your desired Ad5 E1/E3 deleted serotype
- PAC digestion of of purified recombinant BAC DNA
- Virus reconstitution in 293 cells
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