RT Journal Article
SR Electronic
T1 Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.06.22.165704
DO 10.1101/2020.06.22.165704
A1 Daniel Chu
A1 An Nguyen
A1 Spenser S. Smith
A1 Zuzana Vavrušová
A1 Richard A. Schneider
YR 2020
UL http://biorxiv.org/content/early/2020/06/22/2020.06.22.165704.abstract
AB Precisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.