Fundamentals of Baculovirus Expression and Applications.

In 1982 E. coli produced human insulin, the world's first recombinant DNA drug, was approved by the FDA. Since this historical event, remarkable progress has been made in developing bacterial, yeast, mammalian and insect cell protein expression systems that are used to produce recombinant proteins for both research and clinical applications. Of the available approaches, the insect cell based baculovirus expression vector system (BEVS) has proven to be a particularly adaptable system for producing a diverse collection of proteins. Along with E. coli, the system has been valuable for the production of proteins for structural studies, including adequate quantities of difficult to produce G protein-coupled receptors. BEVS has also been used for production of the human papilloma virus vaccine, Cervarix, the first FDA approved insect cell produced product and FluBlok, a vaccine based on the influenza virus hemagglutinin protein. Baculoviruses, modified to contain mammalian promoters (BacMam viruses), have proven to be efficient gene delivery vectors for mammalian cells and provide an alternative transient mammalian cell based protein expression approach to that of plasmid DNA based transfection methodologies. Here we provide an update on recent advances in baculovirus vector development with a focus on the numerous applications of these viruses in basic research and biotechnology.

BacMam-mediated gene delivery into multipotent mesenchymal stromal cells.

Baculoviruses have been used over the last several decades for high-level protein production in insect cells. Recently, modified baculovirus containing a mammalian promoter, known as BacMam virus, has been shown to give high transduction efficiencies across several cell types with minimal cytopathic effects. Cell types amenable to BacMam transduction include primary and adult stem cells. The shuttle vectors used in the construction of BacMam viruses can hold gene fragments up to 38 kb in size, and multiple BacMam viruses can be used in a single transduction for the delivery of more than one gene. BacMam technology has been used in the delivery and expression of targeted fluorescent protein cellular markers, small interfering RNAi, and extensively in the development of cell-based assays. BacMam offers an ideal method for the delivery and expression of large genes in hard-to-transfect cells such as primary and adult stem cells. In this chapter, we describe methods of generating high titer stocks of BacMam for transducing MSC and their derivatives.