Agrobacterium transfection

Marillonnet, S Thoeringer, C., Kandzia, R Klimyuk, V., and Gleba, Y. (2005). Systemic Agrobacterium tumefaciens-mtdiated transfection of viral repli-cons for efficient transient expression in plants. Nat. Biotechnol. 23(6) 718-723. 

Finally, we tested the particle bombardment method, a biolistic approach that was shown before to work when other approaches have failed. In particular, particle bombardment has allowed the generation of transgenic plants such as crop species, which are not susceptible to Agrobacterium tumefaciens or cannot be regenerated from protoplasts. Furthermore, particle bombardment has facilitated organelle transformation in intact cells and the genetic modification of cultured cells that were not accessible to other transfection techniques. 

There exist a variety of vectors for cloning into eukaryotic systems, ranging from yeast (Saccharomyces as well as Pichia) through insect cells (Baculovims) and plants (Ti plasmid from Agrobacterium tumefaciens) to mammalian cells (transfected by viral or mammalian vectors). As expression in eukaryotic hosts is less efficient than bacterial expression in terms of yield and time and more complicated in terms of vector structure and culture conditions, such eukaryotic expression systems are only used for genes whose proteins require posttranslational modification which is not possible in bacteria. Yeast is the preferred option as a relatively easily culturable single-cell system but posttranslational modification capabilities is limited. The additional complexity can be circumvented in part by exploiting the ability of eukaryotic vectors to act as shuttle vectors, which can be shuttled between two evolutionarily different hosts. Thus, eukaryotic vectors can be replicated and analyzed in bacteria and transfected into eukaryotic cells for expression of the recombinant product. 

We recently developed highly active synthetic templates for delivery of RNA viral vectors as DNA precursors using Agrobacterium, and found that Agrobacterium delivery of such templates can be used to start gene amplification and obtain high-level expression in all mature leaves of a plant, simultaneously. Such a transfection route can be performed on an industrial scale by vacuum-infiltration of batches of multiple plants (Marillonnet et al, unpublished results). In this process, the bacteria assume the (formerly viral) functions of primary infection and systemic movement, whereas the viral vector itself provides for cell-to-cell (short distance) spread, amplification and high-level expression. A comparative analysis of state-of-the-art systems with our approach is illustrated in Fig. 6.1. 

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