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BMC Molecular Biology

, 3:13

First Online: 03 September 2002Received: 19 June 2002Accepted: 03 September 2002

Abstract

BackgroundTetracycline-regulated systems have been used to control the expression of heterologous genes in such diverse organisms as yeast, plants, flies and mice. Adaptation of this prokaryotic regulatory system avoids many of the problems inherent in other inducible systems. There have, however, been many reports of difficulties in establishing functioning stable cell lines due to the cytotoxic effects of expressing high levels of the tetracycline transactivator, tTA, from a strong viral promoter.

ResultsHere we report the successful incorporation of tetracycline-mediated gene expression in a mouse mammary epithelial cell line, HC11, in which conventional approaches failed. We generated retroviruses in which tTA expression was controlled by one of three promoters: a synthetic tetracycline responsive promoter TRE, the elongation factor 1-alpha promoter EF1α or the phosphoglycerate kinase-1 promoter PGK, and compared the resulting cell lines to one generated using a cytomegalovirus immediate early gene promoter CMV. In contrast to cells produced using the CMV and PGK promoters, those produced using the EF1α and TRE promoters expressed high levels of β-galactosidase in a tetracycline-dependent manner.

ConclusionsThese novel retroviral vectors performed better than the commercially available system and may have a more general utility in similarly recalcitrant cell lines.

Keywordstetracycline doxycycline inducible expression retroviral vectors transgenics Electronic supplementary materialThe online version of this article doi:10.1186-1471-2199-3-13 contains supplementary material, which is available to authorized users.

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Autor: Paraic A Kenny - Tariq Enver - Alan Ashworth

Fuente: https://link.springer.com/







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