Cytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles results in the efficient generation of germline transgenic mice. Issue 1 (4th December 2015)

• Record Type: Journal Article
• Authors: Garrels, Wiebke; Talluri, Thirumala R.; Ziegler, Maren; Most, Ilka; Forcato, Diego O.; Schmeer, Marco; Schleef, Martin; Ivics, Zoltán; Kues, Wilfried A.
• Is Part Of: Biotechnology journal. Volume 11:Issue 1(2016)
• Page Start: 178
• Page End: 184
• Publication Date: 2015-12-04
• Subjects: Active transgenesis -- Genetic engineering -- Simplified microinjection -- Synthetic biology -- Transposition; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.201500218
• Languages: English
• ISSNs: 1860-6768

Abstract

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. Abstract : Simplified gene transfer method in mice: The cytoplasmic injection of transposon plasmids allows the highly efficient multiplex transgenesis in a single‐step procedure. Due to the transposase‐catalyzed integration of one or more transgenes (here the fluorescent reporters mCherry and Cerulan are depicted by vital imaging of newborn mice; wt, wildtype control), genetically modified mouse models with complex genotypes can be generated in rapid pace.