Stable Recombinase-Mediated Cassette Exchange in Arabidopsis UsingAgrobacterium tumefaciens 

Abstract

Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/ lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycinresistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in highefficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation.

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

“…In this specific implementation of RAGE, we make use of the Cre recombinase and mutated lox site pairings 29,30,[34][35][36] to facilitate integration of large genetic fragments into a precise and predetermined location within the bacterial genome (Fig. 2a).…”

“…Although RAGE offers broad utility for many areas of biology (that is, it is heavily used in genetics studies of higher organisms 29,30 ), the efficiency, flexibility, specificity and speed of recombination make it a disruptive technology for synthetic biology. A detailed schematic of its application to strain implementation is described in Fig.…”

Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

“…A gene conversion approach involving Cre-loxand FLP-FRT-mediated SSI, RMCE, and homologous recombination was explored in maize (Zea mays; Djukanovic et al, 2006). RMCE using two oppositely oriented incompatible lox sites and transiently expressed Cre recombinase produced single-copy RMCE plants in Arabidopsis (Louwerse et al, 2007).…”

Next generation biotechnology: how sophisticated constructs lead to further insights and new approaches towards biotechnology’s demands

“…5 -Fladung et al 2010). Higher cassette exchange frequencies of up to 44% could be shown in Arabidopsis using the Cre/loxP system (Louwerse et al 2007). With the possibility of a site-directed integration or recombinase mediated cassette exchange one can circumvent the extensive work of determining high expression lines for every new construct.…”