Cytoplasmic injection of murine zygotes with Sleeping Beauty transposon plasmids and minicircles results in the efficient generation of germline transgenic mice

• Published in: Biotechnology journal Vol. 11; no. 1; p. 178
• Main Authors: Garrels, Wiebke, Talluri, Thirumala R, Ziegler, Maren, Most, Ilka, Forcato, Diego O, Schmeer, Marco, Schleef, Martin, Ivics, Zoltán, Kues, Wilfried A
• Format: Journal Article
• Language: English
• Published: Germany 01.01.2016
• Subjects: Animals; Cytoplasm - genetics; DNA Transposable Elements - genetics; Gene Transfer Techniques; Genetic Vectors - administration & dosage; Mice; Mice, Transgenic; Microinjections; Plasmids - genetics; Transposases - genetics; Transposases - metabolism; Zygote - growth & development; Simplified microinjection; Transposition; Active transgenesis; Genetic engineering; Synthetic biology
• ISSN: 1860-7314
• DOI: 10.1002/biot.201500218

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.