Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes

Efficient heat-shock removal of the selectable marker gene in genetically modified grapevine

Cisgenesis is one of the new plant breeding technologies emerging as a promising tool for the future, more publicly accepted than the traditional transgenic approach. One of the requirements for a cisgenic plant is the absence of selectable marker genes in the genome. In this study, a system for marker gene removal after selection of transgenic plants has been tested in grapevine. This is based on a binary vector containing a heat-shock-inducible promoter which, upon induction, activates a recombinase to produce the excision of a FRT-flanked box. After the removal of this cassette hosting both the marker gene, nptII, and the recombinase itself, the reporter gene gus may be expressed. Gene transfer experiments on grapevine embryogenic callus were carried out via Agrobacterium tumefaciens. Different heat-shock treatments with variable temperatures and heat incubations times were tested on a selected line and the optimal conditions for a complete removal of nptII with the subsequent gus transcription were found. Plants were analysed by means of qPCR on genomic DNA, to quantify nptII removal, and by a fluorimetric assay to measure gus activity. Our study is conceived as a proof-of-concept to investigate the feasibility of this method in grapevine in view of developing an efficient cisgenic approach in this valuable fruit cro