Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.

Abstract

The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regula- A "genetic switch" that could be operated at will and that would permit the control of individual gene activities quantitatively and reversibly in a temporal and spatial manner would thus be of great advantage. The tetracycline (Tc)-controlled systems for the activation of transcription (7, 8) fulfill a number of these requirements at the cellular level. Herein, we report that the "reverse Tc-controlled transactivator" (rtTA) system, where doxycycline (dox) acts as an inducer of transcription as well as the "Tc-controlled transactivator" (tTA) system, where Tc or dox prevent transcription activation (Fig. 1) can be operated in a quantitative and highly tissue-specific way when transferred into mice. The results show that the controls are tight and that the kinetics of induction, especially with the rtTA system, are rapid. Although we (9, 10) and others (11,12) have reported that the tTA system can be applied to transgenic organisms, the results reported herein establish that both the rtTA and the tTA systems provide true genetic switches capable of quantitatively controlling individual gene activities in animals in a highly tissue-specific manner. These observations open up exciting prospects for the study of gene function in mammalian organisms. Transgenic Animals. Transgenic mouse lines (NMRI outbred) were generated by pronuclear injection using standard techniques (14) and analyzed by the Southern blot technique (15) using the BamHI-EcoRV fragment of the luciferase gene and the XbaI fragment of the tTA gene as respective probes. Luciferase reporter mice were obtained upon transfer of the 3.1-kb XhoI-EaeI fragment of pUHC13-3 (7). Mice producing rtTA controlled by PhCMV, the promoter of the human cytomegalovirus immediate early genes, were obtained upon transfer of the 2.7-kb PflMI-XhoI fragment and animals synthesizing tTA under the control of the LAP promoter were obtained by transferring the 5.5-kbAseI-Asp700 fragment of pUHG15-30. Animals transgenic for both a transactivator and a reporter unit were exposed when necessary to doxycycline hydrochloride (Sigma) dissolved in 5% sucrose supplied as drinking water, which was exchanged every 3 days. Possible long-term effects of dox (200 ,ug/ml) were exam...

Repetitive, non-invasive imaging of the dopamine D2 receptor as a reporter gene in living animals

“…[14][15][16] However, proper synthesis and membrane insertion of the receptor in other tissues, tissue-specific retention of FESP metabolites in non-D 2 R-dependent fashion, and other variables may modify imaging of the D 2 R reporter gene product. To determine carefully lower levels of detection of this reporter gene in vivo, D 2 R reporter gene expression in inducible models [36][37][38] will need to be characterized. It is clear, however, that we can quantitatively determine expression of the D 2 R reporter gene with FESP in vivo, both when the D 2 R is expressed from an infecting viral vector and when the D 2 R is expressed from a stably integrated foreign promoter in heterologous tissue.…”

Cell cycle arrest is sufficient for p53-mediated tumor regression

“…Therefore, we developed an inducible tumor cell line in which wild-type p53 gene expression is controlled by a tetracycline-dependent promoter. 13,14 Using this model, we recently showed that induction of wt-p53 increases the expression of the pro-apoptotic BAX protein in its native, inactive conformation without triggering apoptosis. 14 Thus, we used this model to analyze whether or not the antitumoral activity of wt p53 is dependent on its pro-apoptotic function.…”

In vivo manipulation of interleukin-2 expression by a retroviral tetracycline (tet)-regulated system

“…It is already known that a supply of tet in drinking water leads to a continuous dissemination of the antibiotic in different tissues, whereby tet is readily absorbed and broadly distributed without toxicity at the concentration needed to regulate the activity of the promoter. 10 This was demonstrated for vascularized tissues such as the liver in transgenic mice. 10 We have proven that the antibiotic also easily accesses nonvascularized tissue, such as a depot of tumor cells in a s.c. pocket.…”

“…10 This was demonstrated for vascularized tissues such as the liver in transgenic mice. 10 We have proven that the antibiotic also easily accesses nonvascularized tissue, such as a depot of tumor cells in a s.c. pocket. This assumption derives from the observation that the effect of inhibiting IL-2 expression was readily visible already at the start of tumor growth (i.e., at 10 days after application of the tumor cells and tet, respectively).…”

“…In the presence of the antibiotic, the binding of the tTA to the tetO promoter is impaired, resulting in shutoff of target gene expression in cultured cells [1][2][3][4][5][6] and transgenic mice. [7][8][9][10] In the absence of the antibiotic, expression of the target gene is induced.…”