Figure 2.

Model for recombinase‐mediated cassette exchange (RMCE). The upper linear figure depicts a site in a genomic locus that has previously undergone HR to replace Exon3 with a Neo^R expression cassette flanked by two heterotypic LoxP sites (one a black and one a purple triangle) that cannot recombine with each other. One can then introduce a donor plasmid carrying the cDNA of interest flanked by the same heterotypic LoxP sites (the RMCE exchange cassette plasmid) into the targeted hESCs. Cre recombinase (yellow circles) then catalyzes an exchange between the RMCE exchange cassette plasmid and the targeted locus (black “X”s), removing the antibiotic selection marker and introducing the sequence flanked by the heterotypic LoxP sites into the targeted genomic DNA. Cre can be introduced by using an expression vector or by using Tat‐Cre. In the example shown, a specific point mutation in Exon3 (indicated by the red asterisk) has been introduced into the cell to produce a model for a disease‐initiating mutation. RMCE allows one to introduce various sequences between the heterologous LoxP sites. For example, one can introduce mutations, correct mutations, or, as has been demonstrated, one can introduce all of the components necessary to establish a conditional gene expression system, ¹⁵ giving tremendous research potential to a single targeted cell line.