Cre/loxP, Flp/FRT Systems and Pluripotent Stem Cell Lines

Abstract

Cre and FLP recombinases are the most widely used site-specific recombinases in genome engineering. Both are members of the tyrosine class of recombinases and catalyze the reversible, site-specific recombination between two identical sequences of 34 bp length in the absence of accessory factors. The substrate sequences for Cre and Flp recombinases are called loxP and FRT sites respectively. Cre recombinase was discovered in the E. coli bacteriophage P1 where it plays a crucial role in the life cycle of P1 while Flp recombinase was originally derived from the 2 μ circle of Saccharomyces cerevisiae and catalyzes recombination between inverted repeats within the 2 μ plasmid. This chapter shall provide a brief historical perspective on the discovery and early development of the Cre/loxP and FRT/FLP systems citing key studies that paved the way for the application of these site-specific recombination technologies to the engineering of mammalian genomes. Also included are discussions on the mechanisms of Cre/loxP and FRT/FLP systems application of these site-specific recombinase technologies to the introduction of transgenes in human pluripotent stem cells. Certain studies using mouse pluripotent stem cells will also be discussed in order to highlight the possibility of adopting the same strategy in their human counterparts. Lastly, future prospects for these two site-specific recombinases will be presented.