Dataset Information

Producing a Mammalian GFP Expression Vector Containing Neomycin Resistance Gene.

ABSTRACT: The green fluorescent protein (GFP) was originally isolated from the Jellyfish Aequorea Victoria that fluoresces green when exposed to blue light. GFP protein is composed of 238 amino acids with the molecular mass of 26.9 kD. The GFP gene is frequently used in cellular and molecular biology as a reporter gene. To date, many bacterial, yeast, fungal, plants, fly and mammalian cells, including human, have been created which express GFP. Martin Chalfie, Osamu Shimomura, and Roger Tsien were awarded the 2008 noble prize in chemistry for their discovery and development of GFP. In many studies on mammalian cells, GFP gene is introduced into cells using vector-based systems or a recombinant virus to track the location of a target protein or to study the expression level of the gene of interest, but in these studies there is no selection marker to normalize transfection. According to the importance of neomycin gene as a selection marker in mammalian cells, we aimed to produce a GFP expression vector that contains neomycin gene. GFP gene was separated from pEGFP-N1 vector and was inserted in the back-bone of pCDNA3.1/His/LacZ vector that contained the neomycin gene. The resulted vector contained GFP beside neomycin gene.

SUBMITTER: Izadi M

PROVIDER: S-EPMC3558120 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Producing a Mammalian GFP Expression Vector Containing Neomycin Resistance Gene.

Izadi Manizheh M Abiri Maryam M Keramatipour Mohammad M

Avicenna journal of medical biotechnology 20090401 1

The green fluorescent protein (GFP) was originally isolated from the Jellyfish Aequorea Victoria that fluoresces green when exposed to blue light. GFP protein is composed of 238 amino acids with the molecular mass of 26.9 kD. The GFP gene is frequently used in cellular and molecular biology as a reporter gene. To date, many bacterial, yeast, fungal, plants, fly and mammalian cells, including human, have been created which express GFP. Martin Chalfie, Osamu Shimomura, and Roger Tsien were awarded ...[more]

PMID: 23407141

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Project description:BackgroundPreviously, we showed that aminoglycoside phosphotransferases catalyze the formation of a specific inhibitor of the SWI2/SNF2 proteins. Aminoglycoside phosphotransferases, for example neomycin-resistant genes, are used extensively as selection markers in mammalian transfections as well as in transgenic studies. However, introduction of the neomycin-resistant gene is fraught with variability in gene expression. We hypothesized that the introduction of neomycin-resistant genes into mammalian cells results in inactivation of SWI2/SNF2 proteins thereby leading to global epigenetic changes.MethodologyUsing fluorescence spectroscopy we have shown that the inhibitor, known as Active DNA-dependent ATPase ADomain inhibitor (ADAADi), binds to the SWI2/SNF2 proteins in the absence as well as presence of ATP and DNA. This binding occurs via a specific region known as Motif Ia leading to a conformational change in the SWI2/SNF2 proteins that precludes ATP hydrolysis. ADAADi is produced from a plethora of aminoglycosides including G418 and Streptomycin, two commonly used antibiotics in mammalian cell cultures. Mammalian cells are sensitive to ADAADi; however, cells stably transfected with neomycin-resistant genes are refractory to ADAADi. In resistant cells, endogenous SWI2/SNF2 proteins are inactivated which results in altered histone modifications. Microarray data shows that the changes in the epigenome are reflected in altered gene expression. The microarray data was validated using real-time PCR. Finally, we show that the epigenetic changes are quantized.SignificanceThe use of neomycin-resistant genes revolutionized mammalian transfections even though questions linger about efficacy. In this study, we have demonstrated that selection of neomycin-resistant cells results in survival of only those cells that have undergone epigenetic changes, and therefore, data obtained using these resistant genes as selection markers need to be cautiously evaluated.

Dataset Information

Producing a Mammalian GFP Expression Vector Containing Neomycin Resistance Gene.

Publications

Producing a Mammalian GFP Expression Vector Containing Neomycin Resistance Gene.

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