title: In Vitro Synthesis, Delivery, and Bioavailability of Exogenous mRNA in Gene Transfer Mediated by PiggyBac Transposition.
abstract: Nowadays, nonviral gene transfer is currently of great importance for introducing exogenous genes into genomes and for ensuring that transgene expression is suitable for therapeutic and bioproduction purposes. The piggyBac transposon-based system is particularly interesting since it is easy to engineer and has a large cargo capacity, up to 100 kb. In its setup, the system requires only the piggyBac transposase protein and the transgene delineated by the two piggyBac-specific inverted terminal repeats. Usually the source of transposase is carried by a DNA plasmid. However, the principal drawback of this method is the lasting presence of the transposase, due to episomal persistence or possible integration of the transposase gene vector into the cell's genome. This can lead to genotoxic effects such as multiple genomic integration events and remobilization of the transposon vector once it has been integrated. One alternative to improve the safety of the system is to deliver the transposase as in vitro-synthesized messenger RNA in order to define a very narrow expression window during which a one-shot transposition process would occur. Issues that can be encountered when working on mRNA cell transfer are related to the quality of the synthetic mRNA, the system used to introduce mRNA into the cells and the bioavailability of the mRNA molecules. Here we describe a method to produce mRNA, verify its quality, determine which transfecting reagents can be used and how this mRNA is available to promote the transposition process in HeLa cells. Additionally, we illustrate this method in stromal mesenchymal cell lines in order to support hematopoiesis.
authors.affiliation: LBTM, Institute of Biotechnology, UNIL-EPFL, Station 6, Lausanne, 1015, Switzerland.
authors.forename: Solenne
authors.initials: S
authors.lastname: Bire
authors.affiliation: LNOX, GICC UMR CNRS 7292, UFR de Médecine, Bâtiment Dutrochet, 10 Boulevard Tonnellé, Tours, 37032, France.
authors.forename: Nicole
authors.initials: N
authors.lastname: Ishac
authors.affiliation: LNOX, GICC UMR CNRS 7292, UFR de Médecine, Bâtiment Dutrochet, 10 Boulevard Tonnellé, Tours, 37032, France. florence.bonnin@univ-tours.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Rouleux-Bonnin
mesh_terms: D001682:Biological Availability; D004251:DNA Transposable Elements; D018014:Gene Transfer Techniques; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D012315:RNA Caps / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D019895:Transposases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D012315:RNA Caps; D012333:RNA, Messenger; D019895:Transposases
keywords: ARCA; Bioavailability; Half-life; In vitro transcription; Messenger RNA; Transposition; mRNA trafficking; mRNA transfection; poly(A) tailing