title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.
abstract: Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK.
authors.forename: Maryia
authors.initials: M
authors.lastname: Trubitsyna
authors.affiliation: Institute of Cell Biology, School of Biological Sciences, Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, UK.
authors.forename: Gracjan
authors.initials: G
authors.lastname: Michlewski
authors.affiliation: Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Finnegan
authors.affiliation: Institute of BioEngineering, School of Engineering, University of Edinburgh, Edinburgh EH9 3JL, UK.
authors.forename: Alistair
authors.initials: A
authors.lastname: Elfick
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, UK Centre for Mammalian Synthetic Biology, University of Edinburgh, Edinburgh EH9 3FF, UK.
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Rosser
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Richardson
authors.affiliation: Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3FF, UK.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: French
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005813:Genes, Synthetic; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D055029:Inverted Repeat Sequences; D008055:Lipids / Q000737:chemistry; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014162:Transfection; D019895:Transposases / Q000235:genetics* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D008055:Lipids; C086724:Lipofectamine; C111652:mariner transposases; D019895:Transposases