Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Abstract

StatementWe have designed an optimized and integrating inducible-promoter system to control the timing, spatial domains, and levels of gene misexpression throughout avian development. AbstractPrecisely 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 highresolution gene manipulation in vivo. (2016). Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci Rep 6, 20889. Bellairs, R. and Osmond, M. (2005). The atlas of chick development (2nd edn). Amsterdam ; Boston ; London: Elsevier. Benjamini, Y. and Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal statistical society: series B (Methodological) 57, 289-300. Betancur, P., Bronner-Fraser, M. and Sauka-Spengler, T. (2010). Assembling neural crest regulatory circuits into a gene regulatory network. Annual review of cell and developmental biology 26, 581-603.

Species-specific sensitivity to TGFβ signaling and changes to the Mmp13 promoter underlie avian jaw development and evolution

“…To test if the SNPs adjacent to the RUNX2 binding element play a role in the speciesspecific regulation of the Mmp13 promoter by RUNX2, we transfected chick and duck cells with the -181/184bp Mmp13 promoter constructs and with constructs in which we switched the chick/quail "AG" and duck "CA" SNPs. These same chick and duck cells were also co-transfected with either an empty vector or a dox-inducible Runx2 overexpression construct (Chu et al, 2020). Our results show that in chick cells, Runx2 over-expression significantly decreases the activity of the chick and quail Mmp13 promoter, whereas Runx2 over-expression has little effect on the duck promoter ( Figure 7D).…”

“…To generate Runx2 overexpression constructs, full length cDNA was synthesized using Maxima H Minus first strand cDNA synthesis kit (K1651, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's protocol with 2 μg of total HH37 chick, quail, or duck lower jaw RNA and 100 pmol of d(T)20 VN primer (Chu et al, 2020). The cDNA synthesis reaction was carried out at 50ºC for 30 min, 55ºC for 10 min, 60ºC for 10 min, 65ºC for 10 min, and 85ºC for 5 min.…”

“…Full length Runx2 was amplified by PCR using Q5 Hot Start High-Fidelity DNA Polymerase and cloned using CloneJET PCR Cloning Kit. Full length Runx2 was confirmed by Sanger sequencing and cloned into our pPIDNB custom-made plasmid (Chu et al, 2020), which was digested with AflII (R0520S, NEB, Ipswich, MA, USA) and PstI (R3140S, NEB, Ipswich, MA, USA), using NEBuilder HiFi DNA Assembly Master Mix. The pPIDNB plasmid contains a constitutively active mNeongreen (GFP) (Shaner et al, 2013), which serves as a reporter for transfection or electroporation efficiency; and a doxycyline (dox)-inducible (Gossen et al, 1995;Loew et al, 2010;Heinz et al, 2011) mScarlet-I (RFP) (Bindels et al, 2017).…”

“…No change in promoter activity is observed with a combination of rTGFβ1 and TGFβR1 inhibition compared to untreated cells but activity is lower compared to rTGFβ1 treated cells (2.2fold, p < 0.0001). Additionally, to assess the effects of Runx2 on the regulation of the Mmp13 promoter, we co-transfected cells with -2 kb of the duck or quail Mmp13 promoters attached to luciferase, as well as a species-specific Runx2 overexpression construct (Chu et al, 2020). We find that overexpressing duck Runx2 has no effect on the duck Mmp13 promoter, however, overexpressing quail Runx2 induces the activity of the quail Mmp13 promoter by 2.8-fold ( Figure 6C; p < 0.0001).…”

“…Second, chick and quail are more similar in terms of their jaw shape when compared to duck, whereas the chick jaw is more similar to duck in terms of size (Schneider and Helms, 2003;Eames and Schneider, 2008;Mitgutsch et al, 2011;Smith et al, 2015). Third, chick is a longestablished experimental model system with a well annotated genome (Stern, 2005;Lwigale and Schneider, 2008;Sauka-Spengler and Barembaum, 2008;Jheon and Schneider, 2009;Fish and Schneider, 2014a;Abramyan and Richman, 2018;Gammill et al, 2019), which helps in analyzing data from quail and duck (Ealba and Schneider, 2013;Chu et al, 2020).…”