Construction and application of an inducible system for homogenous expression levels in bulk cell lines.

Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BA...

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Autores principales:	Jun Yu, Helena Müller, Sina Hehn, Steffen Koschmieder, Kai Schönig, Wolfgang E Berdel, Hubert Serve, Carsten Müller-Tidow
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2009
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a25c3a7c7f974376bfa517aaaac91254
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spelling	oai:doaj.org-article:a25c3a7c7f974376bfa517aaaac912542021-11-25T06:21:18ZConstruction and application of an inducible system for homogenous expression levels in bulk cell lines.1932-620310.1371/journal.pone.0006445https://doaj.org/article/a25c3a7c7f974376bfa517aaaac912542009-07-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/19649290/?tool=EBIhttps://doaj.org/toc/1932-6203Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BAC (bacterial artificial chromosome) and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein) allows FACS (fluorescence activated cell sorting) and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a beta-catenin-ERalpha fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-beta-catenin-ERalpha vector demonstrated sensitive inducibility by Tet or Dox (doxycycline) in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.Jun YuHelena MüllerSina HehnSteffen KoschmiederKai SchönigWolfgang E BerdelHubert ServeCarsten Müller-TidowPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 4, Iss 7, p e6445 (2009)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jun Yu Helena Müller Sina Hehn Steffen Koschmieder Kai Schönig Wolfgang E Berdel Hubert Serve Carsten Müller-Tidow Construction and application of an inducible system for homogenous expression levels in bulk cell lines.
description	Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BAC (bacterial artificial chromosome) and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein) allows FACS (fluorescence activated cell sorting) and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a beta-catenin-ERalpha fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-beta-catenin-ERalpha vector demonstrated sensitive inducibility by Tet or Dox (doxycycline) in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.
format	article
author	Jun Yu Helena Müller Sina Hehn Steffen Koschmieder Kai Schönig Wolfgang E Berdel Hubert Serve Carsten Müller-Tidow
author_facet	Jun Yu Helena Müller Sina Hehn Steffen Koschmieder Kai Schönig Wolfgang E Berdel Hubert Serve Carsten Müller-Tidow
author_sort	Jun Yu
title	Construction and application of an inducible system for homogenous expression levels in bulk cell lines.
title_short	Construction and application of an inducible system for homogenous expression levels in bulk cell lines.
title_full	Construction and application of an inducible system for homogenous expression levels in bulk cell lines.
title_fullStr	Construction and application of an inducible system for homogenous expression levels in bulk cell lines.
title_full_unstemmed	Construction and application of an inducible system for homogenous expression levels in bulk cell lines.
title_sort	construction and application of an inducible system for homogenous expression levels in bulk cell lines.
publisher	Public Library of Science (PLoS)
publishDate	2009
url	https://doaj.org/article/a25c3a7c7f974376bfa517aaaac91254
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Construction and application of an inducible system for homogenous expression levels in bulk cell lines.

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