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A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

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A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells. / Nalaskowski, Marcus; Ehm, Patrick; Giehler, Susanne; Mayr, Georg W.

in: ANAL BIOCHEM, Jahrgang 428, Nr. 1, 1, 2012, S. 24-27.

Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung

Harvard

Nalaskowski, M, Ehm, P, Giehler, S & Mayr, GW 2012, 'A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.', ANAL BIOCHEM, Jg. 428, Nr. 1, 1, S. 24-27. https://doi.org/10.1016/j.ab.2012.06.001

APA

Nalaskowski, M., Ehm, P., Giehler, S., & Mayr, G. W. (2012). A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells. ANAL BIOCHEM, 428(1), 24-27. [1]. https://doi.org/10.1016/j.ab.2012.06.001

Vancouver

Nalaskowski M, Ehm P, Giehler S, Mayr GW. A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells. ANAL BIOCHEM. 2012;428(1):24-27. 1. https://doi.org/10.1016/j.ab.2012.06.001

Bibtex

@article{9ff28fd92f884c10bc8e1cc103b34aa4,
title = "A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.",
abstract = "Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.",
keywords = "Animals, Humans, Blotting, Western, Cell Line, Bacterial Proteins/metabolism, Luminescent Proteins/metabolism, Promoter Regions, Genetic/genetics, Genetic Vectors/genetics, Mammals/*metabolism, Biochemistry/*methods, Cells/*metabolism, Enhancer Elements, Genetic, Green Fluorescent Proteins/*metabolism, Mutagenesis/genetics, Recombinant Proteins/*metabolism, Sequence Deletion/genetics, Animals, Humans, Blotting, Western, Cell Line, Bacterial Proteins/metabolism, Luminescent Proteins/metabolism, Promoter Regions, Genetic/genetics, Genetic Vectors/genetics, Mammals/*metabolism, Biochemistry/*methods, Cells/*metabolism, Enhancer Elements, Genetic, Green Fluorescent Proteins/*metabolism, Mutagenesis/genetics, Recombinant Proteins/*metabolism, Sequence Deletion/genetics",
author = "Marcus Nalaskowski and Patrick Ehm and Susanne Giehler and Mayr, {Georg W.}",
year = "2012",
doi = "10.1016/j.ab.2012.06.001",
language = "English",
volume = "428",
pages = "24--27",
journal = "ANAL BIOCHEM",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "1",

}

RIS

TY - JOUR

T1 - A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

AU - Nalaskowski, Marcus

AU - Ehm, Patrick

AU - Giehler, Susanne

AU - Mayr, Georg W.

PY - 2012

Y1 - 2012

N2 - Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

AB - Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

KW - Animals

KW - Humans

KW - Blotting, Western

KW - Cell Line

KW - Bacterial Proteins/metabolism

KW - Luminescent Proteins/metabolism

KW - Promoter Regions, Genetic/genetics

KW - Genetic Vectors/genetics

KW - Mammals/metabolism

KW - Biochemistry/methods

KW - Cells/metabolism

KW - Enhancer Elements, Genetic

KW - Green Fluorescent Proteins/metabolism

KW - Mutagenesis/genetics

KW - Recombinant Proteins/metabolism

KW - Sequence Deletion/genetics

KW - Animals

KW - Humans

KW - Blotting, Western

KW - Cell Line

KW - Bacterial Proteins/metabolism

KW - Luminescent Proteins/metabolism

KW - Promoter Regions, Genetic/genetics

KW - Genetic Vectors/genetics

KW - Mammals/metabolism

KW - Biochemistry/methods

KW - Cells/metabolism

KW - Enhancer Elements, Genetic

KW - Green Fluorescent Proteins/metabolism

KW - Mutagenesis/genetics

KW - Recombinant Proteins/metabolism

KW - Sequence Deletion/genetics

U2 - 10.1016/j.ab.2012.06.001

DO - 10.1016/j.ab.2012.06.001

M3 - SCORING: Journal article

VL - 428

SP - 24

EP - 27

JO - ANAL BIOCHEM

JF - ANAL BIOCHEM

SN - 0003-2697

IS - 1

M1 - 1

ER -