Development of Inducible Molecular Switches Based on All-in-One Lentiviral Vectors Equipped with Drug Controlled FLP Recombinase.
Drug-inducible recombination based on flippase (FLP) is frequently used in animal models and in transgenic cell lines to initiate or to abrogate gene expression. Although the system is highly efficient, functional gene analyses depend on the availability of suitable animal models. In contrast, lentiviral vectors are readily available and versatile tools for the transfer of genetic information into a wide variety of target cells, and can be produced at high titer in a timely manner. To combine the advantages of both approaches, we generated a tight, drug-controlled FLP recombinase consisting of a 5' FKBP12 derived conditional destruction domain and a 3' estrogen receptor ligand binding (ERT2) domain. We successfully constructed lentiviral vectors expressing drug-controlled FLP in combination with a fluorescent reporter for recombination of FLP recognition target (FRT) sites located in trans as well as with target alleles located in cis (all-in-one configuration). In this chapter, we describe the design of the drug controlled FLP recombinase, the construction of molecular switches consisting of FLP expressing lentiviral vectors for inducible recombination of target sites located in cis and in trans, as well as the details for the characterization of lentiviral FLP vectors in cell lines.
Institute of Experimental Hematology, OE6960, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany. maetzig.tobias@mh-hannover.de.
Tobias
T
Maetzig
Institute of Experimental Hematology, OE6960, Hannover Medical School, Carl-Neuberg-Straße 1, 30625, Hannover, Germany. schambach.axel@mh-hannover.de.|Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, Boston, MA, USA. schambach.axel@mh-hannover.de.
Axel
A
Schambach
D000818:Animals; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D057915:Drug Substitution / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D011485:Protein Binding; D011960:Receptors, Estrogen / Q000235:genetics; D011995:Recombination, Genetic
D011960:Receptors, Estrogen; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase
Codon optimization; ERT2; FKBP12; FLP recombinase; FRT; Gene transfer; Lentiviral vector