Inducibility of doxycycline-regulated gene in neural and neuroendocrine cells strongly depends on the appropriate choice of a tetracycline-responsive promoter.
Elucidation of the mechanisms underlying specific receptor activation of neural and neuroendocrine cells will require the establishment of cellular systems that permit the regulation of the expression of the protein of interest. In a tetracycline (Tet)-regulated system, the gene encoding the protein of interest is under the control of a Tet promoter and its transcription is activated in the presence of doxycycline (Dox) by the Tet transactivator rtTA. Acceptable inducibility of the gene's expression requires a high level of its expression in the presence of Dox and a minimal basal expression in the absence of Dox. Two Tet promoters are compared here, the original PhCMV*-1 and the second-generation Ptight, with respect to the inducibility of the gene of interest in neuroendocrine and neural cells genetically engineered to express rtTA, namely PC12-Tet-On cells and MB-G-18 cells (mouse brain-derived cells with the phenotype of neuron-restricted precursors). This study demonstrates that the use of Ptight provided a much higher Dox-induced maximal expression in both cell lines, while the basal activities of the two Tet promoters were at similar levels. The additional use of the Tet-controlled silencer (tTS) caused almost complete abrogation of the leakiness of the Ptight promoter and an increase in the inducibility of the regulated gene, but the maximal levels of gene expression driven in the presence of Dox were also markedly reduced.
Klopotowska
Dagmara
D
Department of Experimental Oncology, L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland.
Strzadala
Leon
L
Matuszyk
Janusz
J
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004318:Doxycycline / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D009490:Neurosecretory Systems / Q000187:drug effects* / Q000378:metabolism; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013752:Tetracycline / Q000494:pharmacology*
D017931:DNA Primers; D013752:Tetracycline; D004318:Doxycycline