Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16

Abstract

Continuous regulation is required to maintain a given cell state or to allow it to change in response to the environment. Studies of the mechanisms underlying such regulation have often been hindered by the inability to control gene expression at will. Among the inducible systems available for regulating gene expression in eukaryotes, the tetracycline (tet) regulatable system has distinct advantages. It is highly specific, non-toxic and non-eukaryotic, and consequently does not have pleiotropic effects on host cell genes. Previously this system also had drawbacks, as it did not extinguish gene expression completely, precluding the study of toxic or growth-inhibitory gene products. We report here the development of a facile reversible tetracycline-inducible retroviral system (designated RetroTet-ART) in which activators and repressors together are expressed in cells. Gene expression can now be actively repressed in the absence of tet and induced in the presence of tet, as we have engineered distinct dimerization domains that allow co-expression of homodimeric tet-regulated transactivators and transrepressors in the same cells, without the formation of non-functional heterodimers. Using this system, we show that growth arrest by the cell cycle inhibitor p16 is reversible and dependent on its continuous expression.

Efficient in vitro and in vivo gene regulation of a retrovirally delivered pro-apoptotic factor under the control of the Drosophila HSP70 promoter

“…than one plasmid, 7,8 in others, the induction is irreversible. 9 In most of these methods, since a selectable marker has also to be introduced with the desired expression sequence, clones expressing the transgene in an inducible manner have to be selected.…”

Prolonged and inducible transgene expression in the liver using gutless adenovirus: A potential therapy for liver cancer

“…[35][36][37][38][39][40] Four major systems have been developed, including regulation by the antibiotic tetracycline, the insect steroid ecdysone or its analogs, the antiprogestin mifepristone (RU486), and chemical "dimerizers" such as the immunosuppressant rapamycin and its analogs. [41][42][43] They all involve the drugdependent recruitment of a transcriptional activation domain to a basal promoter driving the gene of interest but differ in the mechanism of recruitment. [41][42][43][44][45] In the mifepristone system, drug-regulated transcription is achieved by fusing a heterologous DNA-binding domain of yeast GAL4 protein and activation domain of VP-16 or NF-B p65 proteins to a mutant human progesterone receptor that is unaffected by endogenous hormones but is activated by synthetic antiprogestins at doses sufficiently low to avoid side-effects in human.…”

“…[41][42][43] They all involve the drugdependent recruitment of a transcriptional activation domain to a basal promoter driving the gene of interest but differ in the mechanism of recruitment. [41][42][43][44][45] In the mifepristone system, drug-regulated transcription is achieved by fusing a heterologous DNA-binding domain of yeast GAL4 protein and activation domain of VP-16 or NF-B p65 proteins to a mutant human progesterone receptor that is unaffected by endogenous hormones but is activated by synthetic antiprogestins at doses sufficiently low to avoid side-effects in human. 45,46 The properties of the mifepristone-regulated system have been investigated in transgenic animals and naked DNA plasmid in muscles.…”

Adeno-associated viral vector-mediated hypoxia-regulated VEGF gene transfer promotes angiogenesis following focal cerebral ischemia in mice

“…Several inducible promoters have been used to control gene expression, such as tetracycline operons, RU 486, edyasone and other inducible systems. [18][19][20][21][22][23][24][25] For the treatment of ischemic diseases, an ideal control would be the expression of angiogenic factors responding to hypoxia/ischemic. Hypoxia-inducible factor-1 (HIF-1) is a protein that accumulates in the tissue under hypoxic/ ischemia conditions.…”