Hier finden Sie wissenschaftliche Publikationen aus den Fraunhofer-Instituten.

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

: Costello, A.; Lao, N.T.; Gallagher, C.; Capella Roca, B.; Julius, L.A.N.; Suda, S.; Ducrée, J.; King, D.; Wagner, R.; Barron, N.; Clynes, M.


Biotechnology journal 14 (2019), Nr.3, Art. 1800219, 10 S.
ISSN: 1860-6768
ISSN: 1860-7314
Fraunhofer FPC@DCU ()

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.