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

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

Biotechnol J. 2018 Jul 10;:e1800219

Authors: Costello A, Lao NT, Gallagher C, Capella-Roca B, Julius LAN, Suda S, Ducrée J, King D, Wagner R, Barron N, Clynes M

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. We 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 demonstrated 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. We 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, we describe numerous strategies to minimize leaky transgene expression and alterations to current miRNA sponge design.

PMID: 29989353 [PubMed – as supplied by publisher]

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