Journal
NUCLEIC ACIDS RESEARCH
Volume 45, Issue 10, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkx113
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Funding
- Darwin Trust PhD Studentship
- Medical Research Council Career Development Award [G10000564]
- Wellcome Trust University Award [085176/Z/08/Z]
- Engineering and Physical Sciences Research Council [EP/J02175X/1]
- Biotechnology and Biological Sciences Research Council [BB/M018040/1]
- Research Council UK, Open Access Fund
- Biotechnology and Biological Sciences Research Council [BB/M018040/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/J02175X/1] Funding Source: researchfish
- Medical Research Council [G1000564] Funding Source: researchfish
- BBSRC [BB/M018040/1] Funding Source: UKRI
- EPSRC [EP/J02175X/1] Funding Source: UKRI
- MRC [G1000564] Funding Source: UKRI
- Wellcome Trust [085176/Z/08/Z] Funding Source: Wellcome Trust
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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-stepmethod 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.
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