Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system.
J Gene Med
; 9(4): 308-18, 2007 Apr.
Article
en En
| MEDLINE
| ID: mdl-17330923
ABSTRACT
BACKGROUND:
Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors.METHODS:
To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)). The resulting fusion proteins were individually tested for their ability to repress transcription of the constitutively active hypoxanthine phosphoribosyltransferase (HPRT) promoter. In addition, compatibility with the commonly used reverse tetracycline-controlled transactivator system (rtTA-system) and responsiveness to the pharmacological effector doxycycline (DOX) were evaluated. Finally, inducibility, effector-dependent promoter activity and the modification of histone H3 and H4 of the active versus the repressed target promoter were determined.RESULTS:
Fusion of the human deacetylase 4 (HDAC4) carboxy-terminal silencing domain to TetR (B/E) resulted in a functional transcriptional repressor. This novel repressor, termed tTS-H4, efficiently reduced the activity of the murine HPRT promoter and a constitutively active human cytomegalovirus (hCMV) minimal promoter. Furthermore, combining tTS-H4 with the rtTA transcriptional activator allowed for grading, turning off and resuming target gene expression over several orders of magnitude without background.CONCLUSIONS:
The tTS-H4 repressor is compatible with the commonly used rtTA transcriptional activation system and is a versatile new tool for tightly and adjustably regulating conditional gene expression.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Represoras
/
Tetraciclina
/
Terapia Genética
/
Regulación de la Expresión Génica
Límite:
Animals
/
Humans
Idioma:
En
Revista:
J Gene Med
Asunto de la revista:
BIOLOGIA MOLECULAR
/
GENETICA MEDICA
Año:
2007
Tipo del documento:
Article
País de afiliación:
Alemania