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Chapter 26. Engineering Embryonic Stem C... > Designing Substrates for Site-Specif... - Pg. 547

26. Engineering Embryonic Stem Cells with Recombinase Systems 547 3. Designing Substrates for Site-Specific Recombination Upon expression, the SSR acts upon two or more recombinase target sites (RTs) that have been introduced into the genome. Designing how the RTs are deployed in the genome is the first step. The design choices are influenced by the objective and also by certain constraints that aVect the eYciency of recombination. For both tyrosine and large serine recombinases, the site of recombination is a short crossover region of hybrid DNA composed of one strand from each of the substrate RTs. Because the recombination product must have complementarity in the crossover regions, and because none of the RTs usually employed for genetic engineering are palindromic in the hybrid region, it follows that RTs have a directionality. 3.1. Characteristics of loxP and FRTs In general, tyrosine recombinases have RTs that are centered on a palindromic recombinase binding site, which flanks the recombination site. For FLP and Cre, these minimal RTs are termed FRT and loxP respectively (Fig. 26.1A). They are both composed of inverted 13 bp binding sites flanking an 8 bp spacer, which includes the 8 or 6 bp hybrid region. For FLP and other 2 micron recombinases, the minimal RT is usually accompanied by an additional 13 bp binding site. This extra binding site appears to convey only a very moderate eVect for genetic engineering (Ringrose et al., 1999) and is usually not employed. Recombination is usually employed to occur between two identical RTs and is inherently reversible (Ennifar et al., 2003; Van Duyne, 2001). Recombination will still occur between RTs that carry limited sequence varia- tions; however, the sequence in the crossover regions must be identical. Conse- quently, two classes of sequence variations can be defined. First, the binding sites can be altered, which will reduce the binding aYnity of the recombinase for the binding site. Because binding of both FLP and Cre to their palindromic RTs is cooperative (Ringrose et al., 1998), it is possible to mutate one half site without greatly aVecting recombination eYciency as long as the other half site remains wild type. The compensatory eVect of cooperativity has been employed to impose asymmetry upon Cre recombination (Albert et al., 1995; Araki et al., 1997). In this strategy, two mutant loxP sites, termed lox66 and lox71, carry mutations in the left or right 13 bp binding sites, respectively (Fig. 26.1A). After recombination, one product RT will contain both wild type binding half sites, and the other will contain both mutant binding half sites (Fig. 26.2A). The double mutant product RT will have a greatly reduced ability to bind Cre, hence the reverse recombination reaction will be disfavored. Second, the crossover region in the spacer can be altered. Because the sequence in the crossover region must be identical for productive recombination, a variety of