Multiplexing RMCE: Versatile Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology

Detection of vulnerable neurons damaged by environmental insults in utero

“…These results demonstrate the power of our reporter system to detect cells in vivo that exhibit aberrant cellular phenotypes elicited by various types of environmental stressors both at early and later stages of brain development. develop a tool for addressing these questions, we modified our HSE-RFP reporter system to enable tracing of the descendant cells with the expression of GFP, using the Flippase-FRT recombination system (13). Using the codon-optimized version of Flippase (FLPo) (14), we generated a plasmid that includes an HSE-FLPo cassette flanked by two LoxP sites (Fig.…”

Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges

“…RMCE is an SSR catalyzed process in which a target cassette flanked by a set of incompatible (non-crossinteracting), so-called "heterospecific" RTs is exchanged for another cassette that is flanked by identical sites. 7,17 For genomic RMCE, the target resides in the host genome, whereas the gene replacement cassette is introduced into the host cell, traditionally as part of a "donor plasmid." The molar excess of the newly introduced cassette supports an effective exchange of the resident cassette (typically present as a single copy).…”

“…5 We will restrict the present review to two prominent members of the first group, Cre ("causes recombination") and Flp ("flippase"), as well as their respective recognition targets, loxP ("locus of crossover in phage P1"), 6 FRT (Flprecombinase target), and a number of site mutants. 7 By catalyzing the insertion, excision, or inversion of specific DNA segments, both recombinases catalyze a variety of processes in the context of the circular genome of the P1 prophage or the yeast 2-μm circle (overview in Fig. 1).…”

Bio-applications Derived from Site-Directed Genome Modification Technologies

“…This technology, referred to as 'recombinase-mediated cassette exchange' (RMCE), includes loxP sites [Cre recombinase (Liu et al 2006a ;Sandhu et al 2011 ) ] or FRT sites [Flp recombinase (Turan et al 2010 ;Roebroek et al 2011 ) , as in the Flp-in TM system developed by Invitrogen, http://products.invitrogen.com/ivgn/product/K601001 ]. To be effective, these recombinases must be used at very high concentrations, which, in the case of Cre, triggers endonucleolytic activity and therefore cellular toxicity.…”