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Enhancement of cell-specific transgene expression from a Tet-Off regulatory system using a transcriptional amplification strategy in the rat brain.

Liu B, Wang S, Brenner M, Paton JF, Kasparov S - J Gene Med (2008)

Bottom Line: This copy was used to drive expression of tTA.Co-transduction with the two types of viral vectors provided doxycycline-regulated transgene expression in a neuron- or astrocyte-specific manner.Our results demonstrate the effectiveness of the transcriptional amplification strategy in developing viral gene delivery systems that combine the advantages of specific cell type targeting and Tet-inducible expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.

ABSTRACT

Background: The Tet-Off system uses a tetracycline-controlled transactivator protein (tTA) and a tetracycline-responsive promoter element (TRE) to regulate expression of a target gene. This system can be used to achieve regulatable transgene expression in specific cell types by employing a cell-specific promoter to drive tTA expression. Wide applications of this attractive approach are, however, hindered by relatively weak transcriptional activity of most cell-specific promoters. We report here the feasibility of using a transcriptional amplification strategy to overcome the problem.

Methods and results: In the developed cell-type-specific, Tet-inducible lentiviral system, two distinct cellular promoters were tested, a human synapsin-1 promoter for neurons and a compact glial fibrillary acidic protein promoter for astroglial cells. Lentiviral vectors were constructed that contained two copies of one or the other of these two promoters. One copy was used to drive the expression of a chimeric transactivator consisting of a part of the transcriptional activation domain of the NF-kappaB p65 protein fused to the DNA-binding domain of the yeast GAL4 protein. The second copy of the cell-specific promoter was modified by introduction of the GAL4 binding sequences at its 5' end. This copy was used to drive expression of tTA. A gene encoding a red fluorescent protein was cloned into another lentiviral vector under transcriptional control of TRE. Co-transduction with the two types of viral vectors provided doxycycline-regulated transgene expression in a neuron- or astrocyte-specific manner. Compared to control viruses without transcriptional amplification, our enhanced systems were approximately 8-fold more potent in cultured neurons and astroglial cells and at least 8- to 12-fold more potent in the rat brain in vivo.

Conclusions: Our results demonstrate the effectiveness of the transcriptional amplification strategy in developing viral gene delivery systems that combine the advantages of specific cell type targeting and Tet-inducible expression.

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Related in: MedlinePlus

Schematic drawing of lentiviral vectors used in this study. LTR, lentiviral long terminal repeat; Tretight, a modified tetracycline-responsive promoter derived from pTRE-tight-DsRed2 (Clontech); DsRed2, red fluorescent protein; 5 × GAL4BS, five tandem GAL4 binding sites; SYN, human synapsin 1 promoter; GfaABC1D, a compact glial fibrillary acidic protein promoter; GAL4p65, a chimeric transactivator consisting of a part of the transactivatin domain of the murine NF-κB p65 protein fused to the DNA-binding domain of the GAL4 protein from yeast; tTA, tetracycline-controlled transactivator protein
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fig01: Schematic drawing of lentiviral vectors used in this study. LTR, lentiviral long terminal repeat; Tretight, a modified tetracycline-responsive promoter derived from pTRE-tight-DsRed2 (Clontech); DsRed2, red fluorescent protein; 5 × GAL4BS, five tandem GAL4 binding sites; SYN, human synapsin 1 promoter; GfaABC1D, a compact glial fibrillary acidic protein promoter; GAL4p65, a chimeric transactivator consisting of a part of the transactivatin domain of the murine NF-κB p65 protein fused to the DNA-binding domain of the GAL4 protein from yeast; tTA, tetracycline-controlled transactivator protein

Mentions: Five self-inactivated HIV-derived LV vectors (Figure 1) were constructed for this study. (1) LV-Tretight-DsRed2, in which the DsRed2 reporter gene is under control of the TRE-tight promoter; (2) LV-1 × SYN-tTA, in which the Tet-Off system transactivator tTA gene is driven by the SYN promoter only; (3) LV-2 × SYN-tTA, a dual expression cassette containing SYN-driven tTA and the SYN-driven GAL4p65 in a single backbone; (4) LV-1 × GfaABC1D-tTA, in which the tTA gene is transcribed by the GfaABC1D promoter only; and (5) LV-2 × GfaABC1D-tTA, a dual expression cassette containing GfaABC1D-driven tTA and the GfaABC1D-driven GAL4p65 in a single backbone. Thus, the combinations of LV-Tretight-DsRed2 + LV-2 × SYN-tTA and LV-Tretight-DsRed2 + LV-2 × GfaABC1D-tTA constitute the TA-enhanced lentiviral neuronal- or glial-specific Tet-regulatable systems. LV-Tretight-DsRed2 + LV-1 × SYN-tTA and LV-1 × GfaABC1D-tTA, without TA, served as controls.


Enhancement of cell-specific transgene expression from a Tet-Off regulatory system using a transcriptional amplification strategy in the rat brain.

Liu B, Wang S, Brenner M, Paton JF, Kasparov S - J Gene Med (2008)

Schematic drawing of lentiviral vectors used in this study. LTR, lentiviral long terminal repeat; Tretight, a modified tetracycline-responsive promoter derived from pTRE-tight-DsRed2 (Clontech); DsRed2, red fluorescent protein; 5 × GAL4BS, five tandem GAL4 binding sites; SYN, human synapsin 1 promoter; GfaABC1D, a compact glial fibrillary acidic protein promoter; GAL4p65, a chimeric transactivator consisting of a part of the transactivatin domain of the murine NF-κB p65 protein fused to the DNA-binding domain of the GAL4 protein from yeast; tTA, tetracycline-controlled transactivator protein
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2962808&req=5

fig01: Schematic drawing of lentiviral vectors used in this study. LTR, lentiviral long terminal repeat; Tretight, a modified tetracycline-responsive promoter derived from pTRE-tight-DsRed2 (Clontech); DsRed2, red fluorescent protein; 5 × GAL4BS, five tandem GAL4 binding sites; SYN, human synapsin 1 promoter; GfaABC1D, a compact glial fibrillary acidic protein promoter; GAL4p65, a chimeric transactivator consisting of a part of the transactivatin domain of the murine NF-κB p65 protein fused to the DNA-binding domain of the GAL4 protein from yeast; tTA, tetracycline-controlled transactivator protein
Mentions: Five self-inactivated HIV-derived LV vectors (Figure 1) were constructed for this study. (1) LV-Tretight-DsRed2, in which the DsRed2 reporter gene is under control of the TRE-tight promoter; (2) LV-1 × SYN-tTA, in which the Tet-Off system transactivator tTA gene is driven by the SYN promoter only; (3) LV-2 × SYN-tTA, a dual expression cassette containing SYN-driven tTA and the SYN-driven GAL4p65 in a single backbone; (4) LV-1 × GfaABC1D-tTA, in which the tTA gene is transcribed by the GfaABC1D promoter only; and (5) LV-2 × GfaABC1D-tTA, a dual expression cassette containing GfaABC1D-driven tTA and the GfaABC1D-driven GAL4p65 in a single backbone. Thus, the combinations of LV-Tretight-DsRed2 + LV-2 × SYN-tTA and LV-Tretight-DsRed2 + LV-2 × GfaABC1D-tTA constitute the TA-enhanced lentiviral neuronal- or glial-specific Tet-regulatable systems. LV-Tretight-DsRed2 + LV-1 × SYN-tTA and LV-1 × GfaABC1D-tTA, without TA, served as controls.

Bottom Line: This copy was used to drive expression of tTA.Co-transduction with the two types of viral vectors provided doxycycline-regulated transgene expression in a neuron- or astrocyte-specific manner.Our results demonstrate the effectiveness of the transcriptional amplification strategy in developing viral gene delivery systems that combine the advantages of specific cell type targeting and Tet-inducible expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Bristol Heart Institute, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.

ABSTRACT

Background: The Tet-Off system uses a tetracycline-controlled transactivator protein (tTA) and a tetracycline-responsive promoter element (TRE) to regulate expression of a target gene. This system can be used to achieve regulatable transgene expression in specific cell types by employing a cell-specific promoter to drive tTA expression. Wide applications of this attractive approach are, however, hindered by relatively weak transcriptional activity of most cell-specific promoters. We report here the feasibility of using a transcriptional amplification strategy to overcome the problem.

Methods and results: In the developed cell-type-specific, Tet-inducible lentiviral system, two distinct cellular promoters were tested, a human synapsin-1 promoter for neurons and a compact glial fibrillary acidic protein promoter for astroglial cells. Lentiviral vectors were constructed that contained two copies of one or the other of these two promoters. One copy was used to drive the expression of a chimeric transactivator consisting of a part of the transcriptional activation domain of the NF-kappaB p65 protein fused to the DNA-binding domain of the yeast GAL4 protein. The second copy of the cell-specific promoter was modified by introduction of the GAL4 binding sequences at its 5' end. This copy was used to drive expression of tTA. A gene encoding a red fluorescent protein was cloned into another lentiviral vector under transcriptional control of TRE. Co-transduction with the two types of viral vectors provided doxycycline-regulated transgene expression in a neuron- or astrocyte-specific manner. Compared to control viruses without transcriptional amplification, our enhanced systems were approximately 8-fold more potent in cultured neurons and astroglial cells and at least 8- to 12-fold more potent in the rat brain in vivo.

Conclusions: Our results demonstrate the effectiveness of the transcriptional amplification strategy in developing viral gene delivery systems that combine the advantages of specific cell type targeting and Tet-inducible expression.

Show MeSH
Related in: MedlinePlus