Tight regulation of transgene expression by tetracycline‐dependent activator and repressor in brain

Abstract

Methods to temporally and spatially regulate gene mutations will provide a powerful strategy to investigate gene function in the brain. To develop these methods, we have established a tightly regulated system for transgene expression in the forebrain using both a tetracycline (Tc)-dependent transcription activator (rtTA) and a repressor (TetR-Kruppel-associated box). In this system, the repressor binds to the Tc-responsive element (TRE) in the absence of doxycycline (Dox), leading to the repression of leaky activation of TRE-mediated transcription caused by weak binding of rtTA to TRE. Upon Dox administration, only the activator binds to TRE and activates transcription. We tested this system in cultured cells by bicistronically expressing both the regulators using an internal ribosome entry site (IRES). In COS-1, HeLa and SHSY5Y cells, leaky transcription activation led by rtTA in the absence of Dox was repressed without decreasing the level of activated transcription in the presence of Dox. Using this system, transgenic mice were produced that express both the regulators using IRES in the forebrain under the control of the aCaMKII promoter and were bred with transgenic mice carrying the TRE-dependent reporter transgene. In reverse transcription-polymerase chain reaction and in situ hybridization analyses of the forebrain in adult double transgenic mice, the treatment of Dox induces reporter mRNA expression, which was not detected before the treatment and after the withdraw of Dox following the treatment. These results indicate that this system allows the tight regulation of transgene expression in a Dox-dependent fashion in the forebrain and will be useful in investigating gene function in the brain.

Molecular Diversity of Connexin and Pannexin Genes in the Retina of the ZebrafishDanio rerio

“…This quantitative expression analysis supported earlier data generated on the basis of conventional RT-PCR technology showing expression of drCx52.6 and drCx55.5 in the retina only (Dermietzel et al 2000;Zoidl et al 2004b). The very low levels detected in other tissues may represent basal constitutive promoter activity as has been described for Cx32 (Bai et al 1993(Bai et al , 1995 or leaky promoter activity (Uchida et al 2006).…”

Upregulation of Calcium/Calmodulin-Dependent Protein Kinase IV Improves Memory Formation and Rescues Memory Loss with Aging

“…In situ hybridization analysis. In situ hybridization was performed as previously described (Uchida et al, 2006) using a DIG-labeled SV40 poly(A) probe.…”

Development of a tightly-regulated tetracycline-dependent transcriptional activator and repressor co-expression system for the strong induction of transgene expression

“…However, the expression of rtTA has been shown to result in leaky Tc-independent expression because rtTA binds weakly to the TRE, even in the absence of Tc (Kistner et al 1996;Forster et al 1999;Freundlieb et al 1999;Baron and Bujard 2000;Wang et al 2000;Uchida et al 2006). Severe problems may result when this rtTA system is used to regulate the expression of toxic proteins or proteins whose low level of expression is enough to affect the function of endogenous proteins.…”

“…Severe problems may result when this rtTA system is used to regulate the expression of toxic proteins or proteins whose low level of expression is enough to affect the function of endogenous proteins. Therefore, in order to reduce this leaky expression, a system was developed in which the Tc-dependent activator (rtTA) and Tc-dependent transcription repressor (TetR-KRAB) are co-expressed from a single vector using an internal ribosome entry site (IRES; Bornkamm et al 2005;Saqr et al 2006;Uchida et al 2006). In this system, only TetR-KRAB binds to the TRE in the absence of Tc and represses leaky rtTA-dependent transcription.…”