Use of green fluorescent protein/Flp recombinase fusion protein and flow cytometric sorting to enrich for cells undergoing Flp-mediated recombination

Abstract

Flp recombinase has been used extensively for in vivo manipulation of eukaryotic DNA at specific sequences designated as FRT sites. We developed a method to use Flp-mediated recombination without the need for drug resistance or metabolic selection of cells in which recombination has occurred. We generated expression plasmids directing expression of fusion proteins consisting of Flp recombinase and green fluorescent protein (GFP) coding sequences. When the plasmids were introduced into K562 cells containing Flp recombinase substrates and transfected cells were selected for by flow cytometric sorting, GFP-positive cells were enriched 5- to 30-fold for Flp- mediated recombination events compared with unsorted cells. These studies demonstrate the usefulness of GFP/Flp recombinase fusion proteins to manipulate chromosomal DNA in vivo without requiring drug resistance or metabolic marker genes.