Leaky Expression of the TET-On System Hinders Control of Endogenous miRNA Abundance

Abstract

With the ability to affect multiple genes and fundamental pathways simultaneously, miRNA engineering of Chinese Hamster Ovary (CHO) cells has significant advantages over single gene expression or repression. Tight control of these molecular triggers is desirable as it could in theory allow on/off or even tunable regulation of desirable cellular phenotypes. The present study investigated the potential of employing a tetracycline inducible (TET-On) system for conditional knockdown of specific miRNAs but encountered several challenges. The authors show a significant reduction in cell proliferation and culture viability when maintained in media supplemented with the TET-On induction agent Doxycycline at concentrations commonly reported. Calculation of a mature miRNA and miRNA sponge mRNA copy number demonstrates that leaky basal transgene expression in the un-induced state, is sufficient for significant miRNA knockdown. This work highlights challenges of the TET-On inducible expression system for controlled manipulation of endogenous miRNAs with two examples; miR-378 and miR-455. The authors suggest a solution involving isolation of highly inducible clones and use a single cell analysis platform to demonstrate the heterogeneity of basal expression and inducibility. Finally, the authors describe numerous strategies to minimize leaky transgene expression and alterations to current miRNA sponge design.

Immunosuppression by Mutated Calreticulin Released from Malignant Cells

“…Indeed, it is conceivable to (in)activate secretory pathways or the subcellular trafficking of receptors by administering biotin or reversible biotin-mimetic molecules at will in a variety of different applications (Terai et al, 2015). The possibility to switch on or off the system rather rapidly differs from other reversible chemical biological tools such as Tet-on and Tet-off systems that respond with a rather long latency and tend to be ''leaky'' (Costello et al, 2019).…”

Caged Cumate Enables Proximity‐Dependent Control Over Gene Expression

“…The highest concentration of cumate (1 mM) provided the most robust response (Figure 3C, D, S5), but significant switching was observed with as little as 5 μM compound (Figure S6). DsRed production was detected in untreated cells, likely due to leaky expression associated with inducible platforms [48–49] . However, the signal turn‐on observed with cumate is sufficient for many imaging experiments.…”

“…DsRed production was detected in untreated cells, likely due to leaky expression associated with inducible platforms. [48][49] However, the signal turn-on observed with cumate is sufficient for many imaging experiments. In cases where a larger dynamic range is necessary, the operator and repressor sequences can be further engineered.…”

Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders

“…While activities of key TFs are expected to be largely conserved between species, direct comparisons have not been made, complicating comparisons between studies using human versus mouse NGN2 overexpression. Moreover, tetracycline-inducible transcriptional activation can exhibit leaky basal transcription in an uninduced state, potentially compromising iPSC pluripotency ( Costello et al., 2019 ; Tian et al., 2009 ). Therefore, it is important to select lines with low basal TF expression when establishing iPSC lines transduced with iN factors and to monitor spontaneous differentiation when culturing uninduced iPSCs.…”