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, 4 (3), 323-31

A Transposon-Mediated System for Flexible Control of Transgene Expression in Stem and Progenitor-Derived Lineages


A Transposon-Mediated System for Flexible Control of Transgene Expression in Stem and Progenitor-Derived Lineages

Aslam Abbasi Akhtar et al. Stem Cell Reports.


Precise methods for transgene regulation are important to study signaling pathways and cell lineages in biological systems where gene function is often recycled within and across lineages. We engineered a genetic toolset for flexible transgene regulation in these diverse cellular contexts. Specifically, we created an optimized piggyBac transposon-based system, allowing for the facile generation of stably transduced cell lineages in vivo and in vitro. The system, termed pB-Tet-GOI (piggyBac-transposable tetracycline transactivator-mediated flexible expression of a genetic element of interest), incorporates the latest generation of tetracycline (Tet) transactivator and reverse Tet transactivator variants--along with engineered mutants--in order to provide regulated transgene expression upon addition or removal of doxycycline (dox). Altogether, the flexibility of the system allows for dox-induced, dox-suppressed, dox-resistant (i.e., constitutive), and dox-induced/constitutive regulation of transgenes. This versatile strategy provides reversible temporal regulation of transgenes with robust inducibility and minimal leakiness.


Figure 1
Figure 1
Validating the pB-Tet-GOI System for Transgene Manipulation (A) Schematic of pB-Tet-GOI system for transposon-mediated integration along with inducible and reversible transgene expression. (B–D) Response plasmids utilized for directed differentiation experiments. (E) Western blot analysis of response groups grown with and without dox. (F–K) Immunocytochemical staining for HA (DLX2 epitope tag), GFP (dox reporter), TagBFP2 (constitutive reporter), and NGN2 in HuNPCs nucleofected with indicated response plasmids. Scale bar, 100 μm. (L and M) Quantification of HuNPCs harboring indicated response plasmids and differentiated for 4 days (I) or 2 weeks (J) after dox. Error bars represent mean ± SEM. p < 0.05, ∗∗p < 0.01; n = 3 biological replicates per condition per time point. See also Figure S1.
Figure 2
Figure 2
Transactivator Variants Allow for Flexible Manipulation of Transgene Expression (A) Chart of transactivator variants and expected properties. (B) Schematic of plasmid encoding pB-TRE-Bi-mpClover-P2A-Luc2P. (C) Design of in vitro assay for transactivator variant validation. (D–H) Luciferase assay reveals changes in firefly luminescence in the presence and absence of dox (as noted in panels) for the respective transactivator variants. n = 4 biological replicates per condition per time point. Samples normalized to Renilla. Error bars represent mean ± SEM. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figure S2.
Figure 3
Figure 3
pB-Tet Is Non-leaky, Inducible, and Reversible in the Postnatal Mouse Brain (A) Experimental timeline of dox administration after electroporation. Control mice received no dox administration. (B) Schematic illustrating coronal planes of SVZ and OB imaged for panels (C)–(L1). (C–L) Native unstained GFP and TagBFP2 (pseudocolored red) fluorescence was analyzed in the VZ and OB. Significant GFP is observed only with dox. Scale bar, 200 μm. (M) TagBFP2+ (all electroporated cells) and GFP+ (dox-responsive) cell counts in the OB core. (N) Analysis of the mean fluorescence intensity of TagBFP2+ and GFP+ cells in the OB. SVZ, subventricular zone; Str, striatum; OB, olfactory bulb. Data points represent readings from a single litter used to minimize experimental variation that might result with comparing data across litters. Results are consistent with more than three independent experiments assessing dox activation kinetics in independent litters. See also Figure S3.
Figure 4
Figure 4
Addition of Luciferase into pB-Tet-GOI Allows Non-invasive Bioluminescence Imaging of Transgene Expression (A) Bioluminescence analysis revealed firefly activity in mice that received dox (n = 5 biological replicates per condition). (B) Quantification of total flux (p/s) in mice from (A). (C) Bioluminescence analysis of tTA2-CA alongside rtTA-V10 (n = 3, all littermates). (D) Quantification of total flux (p/s) in mice from (C). Error bars represent mean ± SEM. p < 0.05. See also Figure S4.

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