Bacterial Artificial Chromosome (BAC) Technology
Bacterial artificial chromosomes (BACs) are large, stably-maintained genomic clones with the capacity to propagate large and unstable foreign DNA fragments in Escherichia coli. In recent years, BAC technology has been also adapted to the manipulation of viral genomes and became an effective alternative to traditional genetic engineering of recombinant oncolytic viruses, especially large DNA viruses such as herpes, vaccinia virus and baculoviruses. Besides, this innovative technology also allows for fast and convenient generation of recombinant adenoviruses particles. BAC also enables the traceless introduction of any mutation using modern tools of bacterial genetics.
Advantages of BAC Technology
BAC has been a promising tool that should greatly facilitate our ability to manipulate gene expression in oncolytic virus construction. It showed many advantages compared with traditional cloning vectors.
- BACs can accept transgenes up to 300 kb in length, which is sufficient to allow the genomes of oncolytic viruses to be maintained. What's more, the large size of BAC helps to minimize site of integration effects.
- Endogenous gene expression more accurately than other cloning systems.
- BAC maintains stability and high fidelity of insert propagation over multiple generations.
- BAC clones permit an assessment to be made not only of the genomic variation that exists within a virus isolate, but also how these differences impact on viral biology.
- BAC clone helps ameliorate the effects of in vitro passage of virus isolates.
- BAC can be transfected and expressed in numerous mammalian cell lines even if transfection efficiency and copy numbers are low.
BAC Technology in Oncolytic Virus Construction
BAC technology has been applied in the fast and convenient construction of recombinant viruses. Here, we describe the use of BAC cloning to realize genetic manipulation and the generation of recombinant HSV viruses expressing a transgene. Generally speaking, constructing HSV-BACs involves directly inserting a BAC vector into a specific site of the viral genome via homologous recombination or using overlapping cosmids covering the whole genome of HSV to recombine in a eukaryotic cell. Amongst, the former method is more commonly used. For this process, the BAC vector with flanked HSV genomic sequences is linearized using restriction enzymes and co-transfected with purified viral genomic DNA into viral permissive cells.
- Step 1: A BAC vector containing required bacterial genes for replication and maintenance, and homologous flanking sequences (marked in red) is constructed.
- Step 2: Eukaryotic cells are infected with HSV and the circular viral genome is recovered with Hirt extraction.
- Step 3: BAC vector is linearized and viral DNA is co-transfected into eukaryotes and HSV-BAC sequences are recovered.
- Step 4: HSV-BAC DNA is transformed into E. coli. Mutagenesis is performed while viral-BAC is maintained in bacteria. Recombinant viral BAC DNA is extracted.
- Step 5: Viral BAC DNA is transfected into eukaryotes and recombinant viruses are rescued to produce infectious virus.
Fig.1 Construction of viral bacterial artificial chromosomes (BACs). (Nygårdas, 2013)
Applications of BAC Vectors
BAC technology has been widely used to quickly swap in transgenes or create specific mutations more easily than classical homologous recombination during oncolytic virus construction. At Creative Biolabs, we routinely use this technology to create a number of different recombinant viruses bearing either therapeutic or reporter genes. BACs are especially useful for studying large viruses because the DNAs of these viruses are too large to be cloned in individual plasmids. Except for BAC, we also provide other solutions for oncolytic virus construction and engineering.
References
- Nygårdas, M. Herpes simplex virus type 1 (HSV-1) pathogenesis and HSV gene therapy of experimental autoimmune encephalomyelitis. 2013.