Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems

Abstract

MicroRNAs play an essential role in cell homeostasis and have been proposed as therapeutic agents. One strategy to deliver microRNAs is to genetically engineer target cells to express microRNAs of interest. However, to control dosage and timing, as well as to limit potential side-effects, microRNAs’ expression should ideally be under exogenous, inducible control. Conditional expression of miRNA-based short hairpin RNAs (shRNAmirs) via gene regulatory circuits such as the Tet-system is therefore a promising strategy to control shRNAmirs’ expression in research and therapy. Single vector approaches like Tet-On all-in-one designs are more compatible with potential clinical applications by providing the Tet-On system components in a single round of genetic engineering. However, all-in-one systems often come at the expense of heterogeneous and unstable expression. In this study, we aimed to understand the causes that lead to such erratic transgene expression. By using a reporter cell, we found that the degree of heterogeneity mostly correlated with reverse tetracycline transactivator (rtTA) expression levels. Moreover, the targeted integration of a potent rtTA expression cassette into a genomic safe harbor locus functionally rescued previously silenced rtTA-responsive transcription units. Overall, our results suggest that ensuring homogenous and stable rtTA expression is essential for the robust and reliable performance of future Tet-On all-in-one designs.

A Ubiquitous Chromatin Opening Element and DNA Demethylation Facilitate Doxycycline-Controlled Expression during Differentiation and in Transgenic Mice

“…A recent study observed heterogeneity in P tet -controlled expression for so-called “all-in-one” vectors in which the transactivator is expressed from a neighboring constitutive promoter, which could be overcome by increased transactivator expression after targeted integration into the Rosa26 locus. 46 The results of our studies show that the silencing of P tet cassettes during development also occurs if the two transcription units are separated on different chromosomes and even when the transactivator is expressed from the Rosa26 promoter. This may suggest increased silencing pressure during the remodeling of chromatin during differentiation.…”

Uniform transgene activation in Tet-On systems depends on sustained rtTA expression

“…have shown that a lower level of rtTA expression is one of the major causes for non-uniform transgene activation. 19 Here we demonstrated that reduced expression level of rtTA was observed in cells without transgene activation, whereas the supplementation of rtTA expression sufficiently improved both transgene activation and myogenic differentiation in the populations. However, supplementation of rtTA expression is often complicated as it requires additional transgene expressing vector to be introduced in the cells.…”

“…It has been suggested that reduction of rtTA expression is one of the major causes of non-uniform transgene activation of Tet-On and the supplementation of rtTA improves the transgene activation. 19 To investigate whether rtTA expression affects both transgene activation and myogenic differentiation in Tet-MyoD hiPSCs, we attempted to rescue rtTA expression by simultaneously transfecting FMDV-Neo-MYOD1-mCherry vector with an additional CMV enhancer/chicken b-actin (CAG) promoter driving rtTA (CAG-rtTA-EGFP) vector. 23 In the CAG-rtTA-EGFP vector, rtTA is conjugated to EGFP through an encephalomyocarditis virus (EMCV) IRES and its expression is driven by the constitutive CAG promoter.…”

Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro

“…Yet, such a limitation similarly exists for most DNA damage‐mediated model systems (including MMC treatment) as marker‐based sorting would result in populations of few cells which cannot be expanded. Furthermore, the percentage of transgenic cells might in fact be higher than 40%–50% due to limited transactivator levels that might, for example, be triggered by its spontaneous epigenetic silencing (Duran et al, 2022).…”