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

WIEBKE GARRELS; THIRUMALA RAO TALLURI; MAREN ZIEGLER; ILKA MOST; DIEGO FORCATO; MARCO SCHMEER; MARTIN SCHLEEF; ZOLTAN IVICS; WILFRIED A. KUES

Biotechnology Journal

Wiley

Año: 2016 vol. 11 p. 178 - 184

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 o 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.