Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells

Soboleski, Mark R.; Oaks, J. Lindsay; Halford, William P.

doi:10.1096/fj.04-3180fje

Cited by 149 publications

(131 citation statements)

References 49 publications

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“…1C). The use of GFP as a precise and quantitative reporter for gene expression is well documented in the literature, 43,44 and the relative abundances we determined are in good agreement with those determined using epitope tags 45 (correlation coefficient D 0.92). We confirmed our ability to detect changes in chaperone expression using this approach by comparing GFP intensity at 30 C and following heat shock (Ssa, Hsp104, Ydj1 and Sis1 exhibit increased expression after heat shock while Ssb exhibits a modest decrease; not shown).…”

Section: The Rac Antagonizes Induced and Spontaneous Formation Of Thesupporting

confidence: 82%

The ribosome-associated complex antagonizes prion formation in yeast

Amor

Castanzo

Delany

et al. 2015

Prion

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ABSTRACT. The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI C ] prion -an alternative conformer of Sup35 protein -and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Dzuo1 strains. Consistent with this finding, Dzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.

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Section: The Rac Antagonizes Induced and Spontaneous Formation Of Thesupporting

confidence: 82%

The ribosome-associated complex antagonizes prion formation in yeast

Amor

Castanzo

Delany

et al. 2015

Prion

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“…15 Recently, it was demonstrated that GFP also had all the essential properties of a quantitative Lentivirus with enhanced neuron-specific promoters H Hioki et al reporter protein and concluded that GFP is reliable and useful one for analysis of promoter activity by measuring fluorescence intensity. 16 As GFP has the advantage that promoter activity can be easily monitored in single cells in sections, we selected GFP as a reporter protein in the present study.…”

Section: Technical Consideration: Quantification Of Promoter Activitymentioning

confidence: 99%

Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters

et al. 2007

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In the field of basic and clinical neurosciences, it is important to develop a method for easy delivery and persistent expression of transgene in central neurons. We firstly generated lentiviral vectors with five kinds of neuron-specific promoters, such as synapsin I (SYN), calcium/calmodulindependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet-derived growth factor beta chain promoters and then novel hybrid promoters by fusing cytomegalovirus enhancer (E) to those neuron-specific promoters. Neuron-specific expression of green fluorescent protein (GFP) with those promoters was examined in vivo by injecting the lentiviral vectors into the rat neostriatum, thalamus and neocortex. Among all the promoters, SYN promoter displayed the highest specificity for neuronal expression in all the regions examined (more than 96%). Although GFP production by the hybrid promoters was about 2-4 times larger than the non-enhanced promoters, the neuronal specificity was significantly decreased in most cases. However, the neuronal specificity of E/SYN hybrid promoter exhibited the least decrease only in the thalamus. Furthermore, the transcriptional activity and neuronal specificity of E/SYN promoter were sustained for up to 8 weeks. Thus, lentivirus with E/SYN promoter is the best vector for strong persistent expression in neurons.

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“…One decade relative fluorescent unit (RFU) was set as threshold. Data were analyzed with WinMDI (version 2.8) software to obtain the percentage of the GFP positive cells and G mean values (Ducrest et al, 2002;Soboleski et al, 2005).…”

Section: Flow Cytometry Analysismentioning

confidence: 99%

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

et al. 2006

Biotech & Bioengineering

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Viral-mediated gene delivery has been explored for the treatment and protection of cardiomyocytes, but so far there is only one report using cationic polymer for gene delivery to cardiomyocytes in spite of many advantages of polymer-mediated gene delivery. In this study, a cationic poly(b-amino ester) (PDMA) with a degradable backbone and cleavable side chains was synthesized by Michael addition reaction. The toxicity of PDMA to neonatal mouse cardiomyocytes (NMCMs) was significantly lower than that of polyethyleneimine (PEI). PDMA formed stable polyplexes with pEGFP. The dissociation of the polyplexes could be triggered by PDMA degradation, and the dissociation time was tunable via the polymer/pEGFP ratio. In vitro transfection showed that PDMA was an effective and low toxic gene delivery carrier for NMCMs. The PDMA/pEGFP polyplexes transfected EGFP gene to NMCMs with about 28% efficiency and caused little death. In contrast, a significant portion of cardiomyocytes cultured with PEI/pEGFP died.

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Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells

Cited by 149 publications

References 49 publications

The ribosome-associated complex antagonizes prion formation in yeast

The ribosome-associated complex antagonizes prion formation in yeast

Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters

Biodegradable cationic polyester as an efficient carrier for gene delivery to neonatal cardiomyocytes

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