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.
Highlights
Transposon originally identified in salmonid fish[6]
The vector system detailed here further expands the SB toolbox by adding a MultiSite Gateway compatible vector family allowing for highly efficient cloning strategies of extremely complex transgenes from up to four fragments in a directional manner, and a second set of stably integratable reporter vectors for monitoring the activation state of signaling pathways
These highly flexible vectors were developed for rapid cloning and engineering of primary cells and cell lines, providing bulk transgenic cell pools that are usable for experiments after only a few passages for recovery and selection
Summary
The Gaussia luciferase-driving reporter cassette was lifted out via PCR and introduced into pCDNA6/TR-ITR between NotI and BstBI restriction sites. This was done in a multiple step process yielding the pSBTET.reporter vector. Upon reaching 80–90% confluency, 20,000 HEK293 reporter cells can be seeded into 96 wells with three to five replicates depending on the experiment, in 100 μl total volume of culture medium, and subjected to stimulation. To limit stress to the primary hBMSC, selection was raised step-wise: with 40% of total antibiotic concentration given after 24 h, brought to full antibiotic selection 72 h after transfection For constructs containing both a reporter element and an overexpression element, selection was performed using Blasticidin 2 μg/ml in combination with Hygromycin 40 μg/ml. Data from experiments was analyzed using Graphpad Prism Software (GraphPad Software, LaJolla)
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