Gene Expression and Level of Expression

Abstract

Genes encoding heterologous proteins are introduced into the plant genome for several purposes. First, the plant‐made protein can be a tool in fundamental research. Second, the synthesis of heterologous proteins in plants can have biotechnological applications. Plants are capable of producing several types of recombinant proteins. Indeed, for the production of all these proteins, the use of plants is obvious, because they have several important advantages; plants can grow easily and inexpensively in large quantities and they can be harvested and processed with the available agronomic infrastructures and upscaling is very simple. Finally, using processes similar to those used in food and feed industry, specifically desired proteins can be easily purified. However, the stability of accumulation is as important as the accumulation levels themselves. Indeed, the introduced transgenes are not always correctly expressed and are often susceptible to position effects and homology‐dependent gene silencing (HDGS) mechanisms. Although the mechanism that induces silencing is still not completely unravelled, already some factors are defined which influence transgene expression, such as copy number and chromosomal position, methylation pattern in the integration region and properties of the transgene sequence itself.

Low frequency of T‐DNA based activation tagging in Arabidopsis is correlated with methylation of CaMV 35S enhancer sequences

“…Several studies report that the presence of inverted repeats in complex chromosomal structures is the main reason for triggering methylation and subsequent silencing of the introduced sequences [16,20,30,32]. We observed relative high levels of methylation in plants with high numbers of T‐DNA integrations especially when right border inverted repeats are present.…”

Elimination of Transgenic Sequences in Plants by Cre Gene Expression

“…Therefore, single copy transgenic plants are more desirable for commercial practice. Several approaches such as conventional screening amongst a large pool of transformants (De Buck and Depicker, 2004), agrolistics (Hansen and Chilton, 1996), niacinamide application (De Block et al, 1997) or use of Cremediated site-specific recombination (Srivastava et al, 1999) have been developed to select/generate single copy lines. The Cre-lox-based strategy is based on a transgene flanked by lox sites in opposite orientation.…”

High frequency of single‐copy T‐DNA transformants produced by floral dip in CRE‐expressing Arabidopsis plants

“…, 2004). The conventional method for identifying transformants with an elite single‐copy insert is screening of a large pool of transformants by DNA gel‐blot analysis or T‐DNA fingerprinting (De Buck and Depicker, 2004). However, both methods are labour‐intensive and time‐consuming.…”