Construction of a mariner-based transposon for epitope-tagging and genomic targeting.
Mobile genetic elements are often employed for constructing gene fusions or to perform mutagenesis. mariner transposons are well-suited to such applications because of their low site specificity, in vitro activity, and exceptionally broad host range. This report describes a mariner-based method for rapidly creating a large number of insertion mutants that can be converted to in-frame epitope fusions in a single step. First, a mariner-based vector is used to deliver a FLP recombinase substrate randomly into a target molecule. Expression of the FLP recombinase is then induced to catalyse the excision of sequences flanked by FLP recombinase target recognition sites, leaving behind a triple-FLAG epitope. The reversibility of the excision event provides opportunities for using genomic targeting methods easily to create transcriptional or translational fusions to genes of interest.
Chiang
Su L
SL
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, USA.
Rubin
Eric J
EJ
D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D007763:Lac Operon / Q000235:genetics; D016254:Mutagenesis, Insertional; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase