posted on 2021-10-05, 16:14authored byNathalie 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.