A vector platform for the rapid and efficient engineering of stable complex transgenes

Abstract

We describe the generation of a set of plasmid vector tools that allow the rapid generation of complex-interacting stable transgenes in immortalized and primary cells. Of particular importance is inclusion of a mechanism to monitor the activation status of regulatory pathways via a reporter cassette (using Gaussia Luciferase), with control of additional transgene expression through doxycycline de-repression. The resulting vectors can be used to assess regulatory pathway activation and are well suited for regulatory pathway crosstalk studies. The system incorporates MultiSite-Gateway cloning for the rapid generation of vectors allowing flexible choice of promoters and transgenes, and Sleeping Beauty transposase technology for efficient incorporation of multiple transgenes in into host cell DNA. The vectors and a library of compatible Gateway Entry clones are available from the non-profit plasmid repository Addgene.