Use of green fluorescent protein/Flp recombinase fusion protein and flow cytometric sorting to enrich for cells undergoing Flp-mediated recombination.
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.
Sabath
D E
DE
Department of Laboratory Medicine, University of Washington, Seattle 98195-7110, USA. dsabath@u.washington.edu
Shim
M H
MH
D003001:Cloning, Molecular / Q000379:methods*; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D005820:Genetic Testing / Q000379:methods; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007202:Indicators and Reagents / Q000378:metabolism*; D019426:Integrases; D020014:K562 Cells; D008164:Luminescent Proteins / Q000235:genetics*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics; D045522:Recombinases; D011995:Recombination, Genetic / Q000235:genetics*; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D045522:Recombinases; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases; C115006:integron integrase IntI1