Vector Agrobacterium

Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucl Acid Res 12 8711-8721... 

Agrobacterium-mQdi iQd DNA transfer technique and direct gene transfer into protoplasts on the basis of the electroporation technique have frequently been used for the gene transfer methods in plants. However, these methods were hardly adapted to monocotyledonous plants or to varieties in which regeneration system was not established. These deficiencies found in gene transfer techniques will be largely overcome if pollen could be used as a DNA vector. 

The calli used to generate cell lines can be transgenic and express the vaccine or therapeutic protein of interest. Transfer of foreign genes into calli takes place by Agrobacterium-va.Q6M.td transformation, particle bombardment, electroporation of protoplasts, or by viral vectors (Fischer... 

Stiekema, W. I, Heidekamp, F., Louwerse, J. D., Verhoeven, H. A., Dijkhuis, P. (1988). Introduction of foreign genes into potato cultivars Bintje and Desiree using Agrobacterium tumefaciens binary vector. Plant Cell Rep., 7, 47-50. 

Cloning in plants, which makes use of the Ti plasmid vector from Agrobacterium, allows the introduction of new plant traits. 

McBride, K. Summerfelt, K. (1990). Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Molecular Biology 14, 269-76. 

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. 

Scheme 1. Design elements of a typical plant cell expression vector, including promoter sequence (35S), terminator sequence (T7), resistance gene (npt), right and left borders (Br and Bl), and insertion point for genes of interest. This binary vector is suitable for plant and Agrobacterium host cells...

For Agrobacterium transformation, the gene of interest must be carried in a binary vector with appropriate bacterial T-DNA elements. For other transformation methods the requirements maybe less stringent. However, even in these cases, the DNA of interest is typically amplified in bacteria. The choice of a promoter is of particular importance, as this element will dictate both the expression level and the time course of protein expression. The inclusion of additional elements, such as enhancers, 5 and 3 untranslated regions, and fusion elements (e.g. fluorescent or 6-histidine tags), should also be considered for improved expression, detection, and downstream processing. 

The vectors used routinely to produce transgenic plants are derived from the soil bacterium Agrobacterium tumefaciens. In its natural form, this bacterium causes the crown gall disease in which the infected plant produces tumors ( galls ) usually at the base ( crown ) of the plant. 

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