title: Cytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles results in the efficient generation of germline transgenic mice.
abstract: Transgenesis in the mouse is an essential tool for the understanding of gene function and genome organization. Here, we describe a simplified microinjection protocol for efficient germline transgenesis and sustained transgene expression in the mouse model employing binary Sleeping Beauty transposon constructs of different topology. The protocol is based on co-injection of supercoiled plasmids or minicircles, encoding the Sleeping Beauty transposase and a transposon construct, into the cytoplasm of murine zygotes. Importantly, this simplified injection avoids the mechanical penetration of the vulnerable pronuclear membrane, resulting in higher survival rates of treated embryos and a more rapid pace of injections. Upon translation of the transposase, transposase-catalyzed transposition into the genome results in stable transgenic animals carrying monomeric transgenes. In summary, cytoplasmic injection of binary transposon constructs is a feasible, plasmid-based, and simplified microinjection method to generate genetically modified mice. The modular design of the components allows the multiplexing of different transposons, and the generation of multi-transposon transgenic mice in a single step.
authors.affiliation: Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt am Rübenberge, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Garrels
authors.affiliation: Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt am Rübenberge, Germany.
authors.forename: Thirumala R
authors.initials: TR
authors.lastname: Talluri
authors.affiliation: Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt am Rübenberge, Germany.
authors.forename: Maren
authors.initials: M
authors.lastname: Ziegler
authors.affiliation: Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt am Rübenberge, Germany.
authors.forename: Ilka
authors.initials: I
authors.lastname: Most
authors.affiliation: Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt am Rübenberge, Germany.|Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Río Cuarto, Córdoba, Argentina.
authors.forename: Diego O
authors.initials: DO
authors.lastname: Forcato
authors.affiliation: Plasmid Factory GmbH KG, Bielefeld, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Schmeer
authors.affiliation: Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt am Rübenberge, Germany.|Plasmid Factory GmbH KG, Bielefeld, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Schleef
authors.affiliation: Division of Medical Biotechnology, Paul-Ehrlich-Institute, Langen, Germany.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
authors.affiliation: Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt am Rübenberge, Germany. wilfried.kues@fli.bund.de.
authors.forename: Wilfried A
authors.initials: WA
authors.lastname: Kues
mesh_terms: D000818:Animals; D003593:Cytoplasm / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage*; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D010957:Plasmids / Q000235:genetics; D019895:Transposases / Q000235:genetics* / Q000378:metabolism; D015053:Zygote / Q000254:growth & development*
chemical_list: D004251:DNA Transposable Elements; D019895:Transposases; C494471:sleeping beauty transposase, human
keywords: Active transgenesis; Genetic engineering; Simplified microinjection; Synthetic biology; Transposition