Elimination of Transgenic Sequences in Plants by Cre Gene Expression

Abstract

The ability to insert foreign DNA into plant cells opened plenty opportunities for the development of new cell lines and improved varieties for agronomic and industrial purposes. Despite the great advances reached there are still some limitations in plant biotechnology based on genetic transformation. In most cases precise engineering of target genomic loci is difficult. Random DNA integration and multi-copy transgene insertions might result in unpredictable expression or gene silencing. Furthermore, commercial application of plant biotechnology products rises numerous regulatory and biosafety concerns about possible spread of the transgenes into the environment or the presence of selectable marker genes. One of the molecular tools that can help to overcome these limitations is site-specific recombination. Several site-specific recombination systems have been shown to be functional in plant cells: the Cre-lox system from bactreiophage P1 (Dale and Ow, 1990; Odell et al., 1990, Bayley et al., 1992), the FLP-FRT system from Saccharomyces cerevisiae (Lyznik et al., 1993; Lloyd and Davis, 1994; Kilby et al., 1995), the R-RS system from Zygosaccharomyces rouxii (Onouchi et al., 1991), the Gin-gix system from bacteriophage Mu (Maeser and Kahmann, 1991), the CinH-RS2 system from Acetinetobacter (Moon et al., 2011), the ParA system from a plasmid operon parCBA (Thomson et al., 2009) and the Streptomyces phage phiC31 system (Kittiwongwattana et al., 2007, Rubtsova et al., 2008). Currently, Crelox has become the most commonly employed site-specific recombination system. Although both types of recombination catalyzed by the Cre protein, site-specific integration and excision, found practical application (Ow, 2002; Gilbertson, 2003; Lyznik et al., 2003; Gidoni et al., 2008; Wang et al., 2011), the removal of lox-flanked sequences is the most widely used applications of Cre recombinase. The following technologies are based on excisional recombination: (i) regulation of gene expression, (ii) resolution of complex insertion sites to single copy structures, (iii) biological confinement, and (iv) elimination of selectable marker genes. Here we review the progress in the employment of Cre-mediated site-specific excisional recombination for applied plant biology and discuss in detail the advantages, limitations and potential improvements of technologies utilizing the Cre-lox system.