Transient Expression Using Viral Vectors

Compared with whole plants, there has been limited development of foreign protein expression systems specifically for use in tissue culture. Some modifications of expression constructs have resulted in improved protein accumulation or have allowed simplified protein recovery. However, in general, modified expression systems have been tested only in a restricted number of cases and have not resulted in the large increases in product yield required for plant cultures to compete with other foreign protein production vehicles. Transient expression techniques, for example using viral vectors, that have been developed for use in whole plants have not yet been applied in plant tissue culture. 

Upon transfection of a vector into a cell, two options exist for expression. The protein product may be expressed either transiently or stably. Transient transfection is a rapid protein expression technique that may last from several days to approximately 2 weeks with the maximum level of production occurring anywhere from 12 to 96 h posttransfection. When using plasmids, the plasmid and the resulting encoded protein are rapidly reproduced by the cell, then over time, the DNA and its resultant expression disappear. Viral vectors are typically transient in nature in which the expression vector takes control of the cell s protein expression machinery for a limited period prior to cell death and eventual lysis. Transient expression is a convenient method used to produce milligram quantities of protein quickly for experiments and to... 

Transfection is the process of introducing DNA or RNA into eukaryotic ceils. The use of transfection is to study the role and regulation of proteins or to understand the mechanisms of a pathway. Transfection can be transient for rapid analysis or stable , mostly for induction of expression. There are various methods of transfection which include electroporation, viral vectors, DEAE-Dextran, calcium phosphate or Lipofectamine. The choice of transfection depends on the cell type used. The most desirable technique is the one which gives high efficiency of nucleic acid transfection with less interference to the cells physiology and high reproducibility. 

There are two general approaches to cDNA expression, transient expression and stable expression. Transient expression systems are typically based on a viral vector the host cells are infected with cDNA-bearing virus and the cDNA-derived protein is produced. At some point a maximum level of cDNA-derived protein expression is obtained and the protein is harvested for use in incubations. Viral vectors often have cytopathic effects on the host cells which usually precludes analysis of xenobiotic-induced toxicity to the host cell. Stable expression systems can be based on integrating or episomal vectors. With both stable expression approaches, homogeneous, clonally derived populations of cells stably expressing the cDNA are identified and characterized. The properties, advantages and disadvantages of the two approaches are discussed below. 

Table 4 Transient expression of heterologous genes in plant tissues using Agrobacterium-delivered T-DNA (no viral vector)...

Kentucky). The company uses a TMV-based transient expression platform that is fast and easy to apply. The platform is built on viral vectors capable of systemic movement. 

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