title: Genetic engineering of mammalian cells by direct delivery of FLP recombinase protein.
abstract: Protein transduction is based on the ability of certain peptides, designated as cell penetrating peptides (CPPs), to intracellularly deliver cargo molecules, such as peptides and proteins. In combination with site specific recombination, CPP-mediated delivery of recombinases enables a precise and highly efficient control of gene expression in cultured cells and mice. Herein, we provide detailed protocols for engineering and purification of a cell-permeant FLP recombinase protein. Two examples describe the use of cell permeant FLP for excising prespecified fragments from transgenes expressed in fibroblasts and mouse embryonic stem cells. A third example describes the combined use of cell-permeant Cre and FLP recombinases to reversibly induce transgenes in embryonic stem cells. We anticipate that the protocols described herein will be widely used for various genetic interventions addressing complex biological questions.
authors.affiliation: Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn - Life & Brain Center and Hertie Foundation, Sigmund-Freud Straße 25, 53105 Bonn, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Patsch
authors.forename: Daniela
authors.initials: D
authors.lastname: Kesseler
authors.forename: Frank
authors.initials: F
authors.lastname: Edenhofer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018929:Cell Culture Techniques; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016503:Drug Delivery Systems; D053595:Embryonic Stem Cells / Q000378:metabolism*; D004926:Escherichia coli; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D019076:Transgenes
chemical_list: D057846:Cell-Penetrating Peptides; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; C045073:Cre recombinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase; D019426:Integrases