Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 22, Issue 10, Pages -Publisher
MDPI
DOI: 10.3390/ijms22105206
Keywords
gene regulation; expression systems; Tet-On; ribosomal proteins; uL10 (P0)
Funding
- National Science Center in Poland [2015/17/B/NZ1/01777, 2018/02/X/NZ1/00511, UMO-2018/29/B/NZ1/01728]
- Institute of Biological Sciences, Maria Curie-Sklodowska University, Lublin, Poland
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This study presents an optimized inducible expression system that enables rapid generation of doxycycline-responsive cells. The modifications of the Tet-On system provide high, stable, and homogenous expression of the Tet-On3G transactivator.
Precise analysis of the genetic expression and functioning of proteins requires experimental approaches that, among others, enable tight control of gene expression at the transcriptional level. Doxycycline-induced Tet-On/Tet-Off expression systems provide such an opportunity, and are frequently used to regulate the activity of genes in eukaryotic cells. Since its development, the Tet-system has evolved tight gene control in mammalian cells; however, some challenges are still unaddressed. In the current set up, the establishment of the standard Tet-based system in target cells is time-consuming and laborious and has been shown to be inefficient, especially in a long-term perspective. In this work, we present an optimized inducible expression system, which enables rapid generation of doxycycline-responsive cells according to a one- or two-step protocol. The reported modifications of the Tet-On system expand the toolbox for regulated mammalian gene expression and provide high, stable, and homogenous expression of the Tet-On3G transactivator, which is of fundamental importance in the regulation of transgenes.
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