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The Tetracycline-Controlled Transactivator (Tet-On/Off) System in Beta Cells Reduces Insulin Expression and Secretion in Mice

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posted on 05.10.2021, 16:14 by Nathalie Jouvet, Khalil Bouyakdan, Scott A. Campbell, Cindy Baldwin, Shannon E. Townsend, Maureen A. Gannon, Vincent Poitout, Thierry Alquier, Jennifer L. Estall
Controllable genetic manipulation is an indispensable tool in research, greatly advancing our understanding of cell biology and physiology. However in beta cells, transgene silencing, low inducibility, ectopic expression and off-targets effects are persistent challenges. In this study, we investigated whether an inducible, Tet-Off system with beta-cell specific MIP-itTA driven expression of TetO-CreJaw/J could circumvent previous issues of specificity and efficacy. Following assessment of tissue-specific gene recombination; beta cell architecture; in vitro and in vivo glucose-stimulated insulin secretion; and whole-body glucose homeostasis, we discovered that expression of any tetracycline-controlled transactivator (e.g. itTA, rtTA or tTA) in beta cells significantly reduced Insulin gene expression and decreased insulin content. This translated into lower pancreatic insulin levels and reduced insulin secretion in mice carrying any tTA transgene, independent of Cre recombinase expression or doxycycline exposure. Our study echoes ongoing challenges faced by fundamental researchers working with beta cells and highlights the need for consistent and comprehensive controls when using the Tet-On or Tet-Off systems for genome editing.

Funding

Supported by operating funds to J.L.E. from the CIHR (PJT-148771), the Montreal Diabetes Research Center (MDRC) and Diabetes Quebec, to V.P. by funds from the National Institutes of Health (R01-DK58096) and Canadian Institutes of Health Research (grant MOP 77686), to M.A.G. by funds from the Department of Veterans Affairs Merit review (1IO1-BX0037440-01) and NIH/NIDDK (R01-DK120626) and to T.A. by funds from CIHR (PJT-153035). S.E.T. is supported by the Vanderbilt University Training Program in Molecular Endocrinology (5T32 DK7563-30). J.L.E. and T.A. are supported by salary awards from the FRQS.

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