Recombinant Adenovirus Construction in Bacterial Systems
Adenoviral (Ad) vectors have been treated as a versatile tool in the investigations of gene expression and regulation as well as gene therapy. During the past years, many approaches have been developed for the construction of recombinant adenoviruses. Among which a homologous recombination system based on bacterial systems, such as Escherichia coli, has been developed to generate Ad vectors. With years of experience and abundant experience, Creative Biolabs is capable of providing high-quality recombinant Ad vector construction using bacterial systems.
Recombinant Ad Vectors Construction in Bacterial System
The bacterial system based recombinant Ad vector construction is an indirect method which utilizes two plasmids coding for the homologous recombinant regions: one is a shuttle plasmid containing an expression cassette, and the other is the large plasmid containing the majority of the adenoviral genome. With this method, the vector plasmid and the shuttle plasmid are cotransformed with recBCsbcBC E. coli. The recombination event occurs through the overlapping of the fragments of each plasmid. Then, the plasmid is isolated after culturing of the independent E. coli clones for a short time. Since recBCsbcBC E. coli is not suitable for large-scale preparation of the plasmid, the recovered plasmid is retransformed and cultured with more conventionally used strains of E. coli (e.g., DH5α). The plasmid is then isolated, and positive clones are selected by restriction analysis.
Figure 1. Schematic representation of the AdEasy technology. (Luo, 2007)
Features of Bacterial System-Based Recombinant Ad Vectors Construction
Different from the traditional Ad vector construction method which is time-consuming, the bacterial system-based method allows the rapid and efficient cloning and manipulation of full-length infectious adenovirus genomes in bacterial plasmids. It also combines the powerful genetic engineering techniques that are available in E. coli and the ability of this microorganism to recombine homologous sequences at a high frequency. What's more, this method has many advantages, which are characterized by:
- High frequency of bacterial colonies containing the plasmid with the modified adenovirus genome
- Viral genome can be specifically modified or deleted with appropriate restriction sites
- Full-length adenovirus genomes can be introduced into appropriate bacterial strains for production of large amounts of viral DNA
As a frontier service provider in gene therapy, Creative Biolabs is dedicated to offering the best-characterized recombinant adenovirus construction services for our global clients. Our professional scientists will select the most appropriate system and advanced techniques to meet your specific projects. Please contact us for more detailed information and our team will get back to you as soon as possible.
Reference
- Luo, J.; et al. (2007). A protocol for rapid generation of recombinant adenoviruses using the AdEasy system. Nature protocols. 2(5): p.1236.
