title: piggyBac transposon plus insulators overcome epigenetic silencing to provide for stable signaling pathway reporter cell lines.
abstract: Genetically modified hematopoietic progenitors represent an important testing platform for a variety of cell-based therapies, pharmaceuticals, diagnostics and other applications. Stable expression of a transfected gene of interest in the cells is often obstructed by its silencing. DNA transposons offer an attractive non-viral alternative of transgene integration into the host genome, but their broad applicability to leukocytes and other "transgene unfriendly" cells has not been fully demonstrated. Here we assess stability of piggyBac transposon-based reporter expression in murine prostate adenocarcinoma TRAMP-C2, human monocyte THP-1 and erythroleukemia K562 cell lines, along with macrophages and dendritic cells (DCs) that have differentiated from the THP-1 transfects. The most efficient and stable reporter activity was observed for combinations of the transposon inverted terminal repeats and one 5'- or two cHS4 core insulators flanking a green fluorescent protein reporter construct, with no detectable silencing over 10 months of continuous cell culture in absence of any selective pressure. In monocytic THP-1 cells, the functional activity of luciferase reporters for NF-κB, Nrf2, or HIF-1α has not decreased over time and was retained following differentiation into macrophages and DCs, as well. These results imply pB as a versatile tool for gene integration in monocytic cells in general, and as a convenient access route to DC-based signaling pathway reporters suitable for high-throughput assays, in particular.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, Missouri, United States of America ; Experiment Station Chemical Labs, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Valeri V
authors.initials: VV
authors.lastname: Mossine
authors.affiliation: Experiment Station Chemical Labs, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: James K
authors.initials: JK
authors.lastname: Waters
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Mark
authors.initials: M
authors.lastname: Hannink
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia, Missouri, United States of America ; Experiment Station Chemical Labs, University of Missouri, Columbia, Missouri, United States of America ; Department of Child Health, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Mawhinney
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004251:DNA Transposable Elements / Q000235:genetics*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008156:Luciferases; D051379:Mice
chemical_list: D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; D008156:Luciferases