A vector platform for the rapid and efficient engineering of stable complex transgenes.
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.
Medizinische Klinik und Poliklinik IV, Klinikum Innenstadt, Ludwig-Maximilians-University Munich, Clinical Biochemistry Group, Munich, Germany.
Carsten
C
Jäckel
Medizinische Klinik und Poliklinik IV, Klinikum Innenstadt, Ludwig-Maximilians-University Munich, Clinical Biochemistry Group, Munich, Germany.
Melanie Schmitt
MS
Nogueira
Medizinische Klinik und Poliklinik IV, Klinikum Innenstadt, Ludwig-Maximilians-University Munich, Clinical Biochemistry Group, Munich, Germany.
Nadja
N
Ehni
Medizinische Klinik und Poliklinik IV, Klinikum Innenstadt, Ludwig-Maximilians-University Munich, Clinical Biochemistry Group, Munich, Germany.
Christiane
C
Kraus
Medizinische Klinik und Poliklinik IV, Klinikum Innenstadt, Ludwig-Maximilians-University Munich, Clinical Biochemistry Group, Munich, Germany.
Julius
J
Ranke
Medizinische Klinik und Poliklinik IV, Klinikum Innenstadt, Ludwig-Maximilians-University Munich, Clinical Biochemistry Group, Munich, Germany.
Maike
M
Dohmann
Helmholtz Zentrum München Immunoanalytics- Core Facility, Großhadern, Germany.
Elfriede
E
Noessner
Medizinische Klinik und Poliklinik IV, Klinikum Innenstadt, Ludwig-Maximilians-University Munich, Clinical Biochemistry Group, Munich, Germany.
Peter J
PJ
Nelson
D003001:Cloning, Molecular; D004318:Doxycycline / Q000494:pharmacology; D005818:Genetic Engineering; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D019076:Transgenes
D004318:Doxycycline
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