Ecdysone Receptor-based Singular Gene Switches for Regulated Transgene Expression in Cells and Adult Rodent Tissues

Abstract

Controlled gene expression is an indispensable technique in biomedical research. Here, we report a convenient, straightforward, and reliable way to induce expression of a gene of interest with negligible background expression compared to the most widely used tetracycline (Tet)-regulated system. Exploiting a Drosophila ecdysone receptor (EcR)-based gene regulatory system, we generated nonviral and adenoviral singular vectors designated as pEUI(+) and pENTR-EUI, respectively, which contain all the required elements to guarantee regulated transgene expression (GAL4-miniVP16-EcR, termed GvEcR hereafter, and 10 tandem repeats of an upstream activation sequence promoter followed by a multiple cloning site). Through the transient and stable transfection of mammalian cell lines with reporter genes, we validated that tebufenozide, an ecdysone agonist, reversibly induced gene expression, in a dose- and time-dependent manner, with negligible background expression. In addition, we created an adenovirus derived from the pENTR-EUI vector that readily infected not only cultured cells but also rodent tissues and was sensitive to tebufenozide treatment for regulated transgene expression. These results suggest that EcR-based singular gene regulatory switches would be convenient tools for the induction of gene expression in cells and tissues in a tightly controlled fashion.

IQ-Switch is a QF-based innocuous, silencing-free, and inducible gene switch system in zebrafish

“…1e ). The relatively slow off-kinetics of Teb corresponded well with the previous reports 4 , 23 , 29 showing that it took time to completely metabolize ecdysteroids and their agonists in cells. In mice, the ecdysteroids are taken up and metabolized by the liver before excretion through both urinary and fecal routes 23 .…”

“…Although there are miscellaneous binary transgene induction cassettes available, one or more inherent weaknesses have hampered their application for the generation of transgenic animals. For instance, Tet-On/Off and stress-inducible promoters are not recommended for conditional gene expression due to the relatively high leakiness of the transgene 4 , 41 . The GAL4/UAS system has been most successfully applied to zebrafish in transgenesis as well as in enhancer trapping 42 , 43 owing to its many advantages including a tolerable degree of driver toxicity, low leakiness, reversibility, and a relatively high expression level of transgenes.…”

“…Therefore, it would be desirable to substitute Teb with other ecdysone analogs with less or no cellular toxicity. In addition, for expanding IQ-Switch in broader biological applications, generating a singular plasmid by combining all the requisite components of the driver and effector of IQ-Switch would be helpful, as was the case with a Gal4/UAS-based singular gene switch 4 .…”

“…Especially, a sophisticatedly designed heterologous GAL4-TA composed of an isolated GAL4 DNA-binding domain and a minimal VP16 activation domain fused to a modified ecdysone-binding region of EcR had been adopted in zebrafish for the controlled expression of transgenes which could be tightly regulated by several different ecdysone agonists 3 . However, the relatively high leakiness of Tet-On/Off 4 and the silencing of GAL4-responsive UAS element due to accumulated methylations on its CpG dinucleotides in vertebrates 5 have been potentially problematic the use of these systems in transgenesis. The leakiness of ITESs could be completely plugged by concomitantly exploiting inducible Flp/ FRT or Cre/ LoxP DNA recombinase-based systems to initiate transgene expression by the excision of regulatory elements 6 , 7 .…”

Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies

“…US28 constructs with N-terminal NLuc were generated by replacing CXCR4 with US28 in the NLuc-CXCR4 pcDNA3.1 (+) construct, which was described previously 65 , and subcloning it to the pcDEF3 vector. FLAG-tagged intrabodies were generated by subcloning nanobodies with a C-terminal FLAG tag to the pEUI(+) vector containing the ecdysone-inducible expression system 66 . Plasmids for lentiviral transduction were generated by subcloning US28 ICL2 CCR5 and intrabody constructs from the pcDEF3 vector to the pLenti6.3/To/V5-DEST vector.…”