For most experimental and therapeutic applications of gene transfer, regulation of the timing and level of gene expression is preferable to constitutive gene expression. Among the systems that have been developed for pharmacologically controlled gene expression in mammalian cells, the bacterial tetracycline (tet)-responsive system has the advantage that it is dependent on a drug (tet) that is both highly specific and non-toxic. The tet-responsive system has been previously used to modulate expression of cell cycle regulatory proteins in cultured cells, reporter genes in plants and transgenic mice and reporter genes directly injected into the heart. Here we show that orally or parenterally administered tet regulates expression of tet-responsive plasmids injected directly into mouse skeletal muscle. Reporter gene expression was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn. These data show that skeletal muscle offers an accessible and well characterized target tissue for tet-controlled expression of genesin vivo, suggesting applications to developmental studies and gene therapy.
"The addition of doxycycline to the drinking water (500 μg/mL) for the last 4 days caused a robust increase in the expression of eGFP at the site of the tumour, as evidenced by fluorescence imaging on the entire brain removed from the skull (Fig. 2C). Even though a doxycycline concentration of 2 mg/ mL in the drinking water is commonly used (which corresponds to 100 mg/kg in rats) (Dhawan et al., 1995 "
[Show abstract][Hide abstract] ABSTRACT: High grade gliomas are known to release excitotoxic concentrations of glutamate, a process thought to contribute to their malignant phenotype through enhanced autocrine stimulation of their proliferation and destruction of the surrounding nervous tissue. A model of C6 glioma cells in which expression of the high affinity glutamate transporter GLT-1 can be manipulated both in vivo and in vitro was used in order to investigate the consequences of increasing glutamate clearance on tumour progression. These cells were grafted in the striatum of Wistar rats and doxycycline was administered after validation of tumour development by magnetic resonance imaging. Both GLT-1 expression examined by immunohistochemistry and glutamate transport activity measured on synaptosomes appeared robustly increased in samples from doxycycline-treated animals. Moreover, these rats showed extended survival times as compared to vehicle-treated animals, an effect that was consistent with volumetric data revealing delayed tumour growth. As constitutive deficiency in glutamate clearance at the vicinity of brain tumours is well established, these data illustrate the potential benefit that could be obtained by enhancing glutamate transport by glioma cells in order to reduce their invasive behaviour.
[Show abstract][Hide abstract] ABSTRACT: We describe a single autoregulatory cassette that allows reversible induction of transgene expression in response to tetracycline (tet). This cassette contains all of the necessary components previously described by others on two separate plasmids that are introduced sequentially over a period of months [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. The cassette is introduced using a retrovirus, allowing transfer into cell types that are difficult to transfect. Thus, populations of thousands of cells, rather than a few clones, can be isolated and characterized within weeks. To avoid potential interference of the strong retroviral long terminal repeat enhancer and promoter elements with the function of the tet-regulated cytomegalovirus minimal promoter, the vector is self-inactivating, eliminating transcription from the long terminal repeat after infection of target cells. Tandem tet operator sequences and the cytomegalovirus minimal promoter drive expression of a bicistronic mRNA, leading to transcription of the gene of interest (lacZ) and the internal ribosome entry site controlled transactivator (Tet repressor-VP16 fusion protein). In the absence of tet, there is a progressive increase in transactivator by means of an autoregulatory loop, whereas in the presence of tet, gene expression is prevented. Northern blot, biochemical, and single cell analyses have all shown that the construct yields low basal levels of gene expression and induction of one to two orders of magnitude. Thus, the current cassette of the retroviral construct (SIN-RetroTet vector) allows rapid delivery of inducible genes and should have broad applications to cultured cells, transgenic animals, and gene therapy.
Proceedings of the National Academy of Sciences 05/1996; 93(11):5185-90. DOI:10.1073/pnas.93.11.5185 · 9.67 Impact Factor
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