Excision of selectable marker genes from transgenic plants by site-specific DNA recombination

Abstract

Abstract Current genetic transformation methods have relatively low efficiencies, e.g. in the successful model species Arabidopsis thaliana , the transformation efficiency is in the range 0.05 to 5%. This requires the use of selectable markers to identify the transformants among the untransformed cells. As many of the most efficient selectable markers are antibiotic and herbicide resistance genes, which have perceived biosafety concerns, the development of marker-free GM products is likely to be advantageous for public acceptance and compliance with regulatory requirements. To date, three approaches have been employed to generate marker-free transgenic plants. One approach involves removal of a selectable marker gene from transgenic plants (marker removal). A second method is to overexpress specific genes that are capable of promoting transgenic plant regeneration thus allowing their preferential regeneration compared with untransformed plants (marker-free transformation). The third method was developed from a combination of these two methods. This review highlights recent advances in the development of site-specific DNA recombinase-based systems for marker removal. A simple system consists of a site-specific DNA recombinase (e.g. Cre, Flp or R) and its corresponding recognition sites ( lox , FRT or RS , respectively). The recombinase specifically recognizes its target site, cleaves the double-stranded DNA and then specifically rejoins certain double DNA ends to complete the entire recombination process. Most of the current marker excision methods are based on this type of site-specific DNA recombination system.