An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells

Zhang, Guohong; Gurtu, Vanessa; Kain, Steven R.

doi:10.1006/bbrc.1996.1573

Cited by 347 publications

(216 citation statements)

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“…Protein samples (10 mg) were separated by reducing SDS-PAGE (12 %) in Criterion cassettes (BioRad) with 26-well combs for 70 min at 200 mV and analysed by quantitative immunoblotting. Each immunoblot included enhanced GFP (E-GFP) (Zhang et al, 1996) purified from recombinant E. coli as a standard (37, 55.5, 74, 92.5, 111, 148 and 185 ng) and was performed with Living Colours Aequorea victoria peptide rabbit immunoglobulin anti-GFP antibody (a-GFP) as the primary antibody (Clontech Laboratories) and horseradish peroxidase-conjugated antirabbit immunoglobulin (H+L) antibody raised in goats (Southern Biotechnology Associates) as the secondary antibody, as previously described . Cultures (25 ml) were also grown (37 uC, 200 r.p.m.)…”

Section: Methodsmentioning

confidence: 99%

Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii

et al. 2010

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Proteolysis is important not only to cell physiology but also to the successful development of biocatalysts. While a wide-variety of signals are known to trigger protein degradation in bacteria and eukaryotes, these mechanisms are poorly understood in archaea, known for their ability to withstand harsh conditions. Here we present a systematic study in which single C-terminal amino acid residues were added to a reporter protein and shown to influence its levels in an archaeal cell. All 20 amino acid residues were examined for their impact on protein levels, using the reporter protein soluble modified red-shifted GFP (smRS-GFP) expressed in the haloarchaeon Haloferax volcanii as a model system. Addition of hydrophobic residues, including Leu, Cys, Met, Phe, Ala, Tyr, Ile and Val, gave the most pronounced reduction in smRS-GFP levels compared with the addition of either neutral or charged hydrophilic residues. In contrast to the altered protein levels, the C-terminal alterations had no influence on smRS-GFP-specific transcript levels, thus revealing that the effect is post-transcriptional.

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Section: Methodsmentioning

confidence: 99%

Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii

et al. 2010

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“…EGFP is a valuable reporter in living cells with a sensitivity comparable with that of common in vitro reporter systems. [9][10][11] In order to assess applicability of a simple EGFP gene construct based on the 'strong' constitutive CMV promoter, the EGFP coding sequence, and the SV40 poly adenylation signal, we analyzed fluorescence in several cell types using intranuclear microinjection of 1 to 10 5 copies of pEGFP-N1 plasmid (Clontech, Palo Alto, CA, USA) per injection volume anticipated to be 1 pl. 12,13 For re-localization and comparability, standardized injection tips (Eppendorf, Hamburg, Germany) were used to inject rows of 100 cells per aequous pEGFP solution (in TE after having established that 0.5 × TAE, PBS, and H 2 O give similar results) containing 1, 10, 10 2 , 10 3 , 10 4 , and 10 5 molecules ( Figure 1).…”

Section: Abstract: Human Artificial Chromosome; Mammalian Artificial mentioning

confidence: 99%

Visible transient expression of EGFP requires intranuclear injection of large copy numbers

Schindelhauer

Laner

2002

Gene Ther

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“…To maximize GFP fluorescence, S65TGFP cDNA was exchanged for enhanced GFP cDNA using AgeI/KpnI. Enhanced GFP is codon-optimized for expression in mammalian systems and exhibits 6-fold greater levels of fluorescence than S65T-GFP (21). DNA sequence analysis of the GFP-CFTR junction confirmed the intended reading frame.…”

Section: Gfp-cftr and Cftr Expression Vectorsmentioning

confidence: 99%

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

Moyer¹,

Loffing²,

Schwiebert³

et al. 1998

Journal of Biological Chemistry

152

170

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The mechanism by which cAMP stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl ؊ ) secretion is cell type-specific. By using Madin-Darby canine kidney (MDCK) type I epithelial cells as a model, we tested the hypothesis that cAMP stimulates Cl ؊ secretion by stimulating CFTR Cl ؊ channel trafficking from an intracellular pool to the apical plasma membrane. To this end, we generated a green fluorescent protein (GFP)-CFTR expression vector in which GFP was linked to the N terminus of CFTR. GFP did not alter CFTR function in whole cell patchclamp or planar lipid bilayer experiments. In stably transfected MDCK type I cells, GFP-CFTR localization was substratum-dependent. In cells grown on glass coverslips, GFP-CFTR was polarized to the basolateral membrane, whereas in cells grown on permeable supports, GFP-CFTR was polarized to the apical membrane. Quantitative confocal fluorescence microscopy and surface biotinylation experiments demonstrated that cAMP did not stimulate detectable GFP-CFTR translocation from an intracellular pool to the apical membrane or regulate GFP-CFTR endocytosis. Disruption of the microtubular cytoskeleton with colchicine did not affect cAMP-stimulated Cl ؊ secretion or GFP-CFTR expression in the apical membrane. We conclude that cAMP stimulates CFTR-mediated Cl ؊ secretion in MDCK type I cells by activating channels resident in the apical plasma membrane.

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An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells

Cited by 347 publications

References 1 publication

Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii

Hydrophobic carboxy-terminal residues dramatically reduce protein levels in the haloarchaeon Haloferax volcanii

Visible transient expression of EGFP requires intranuclear injection of large copy numbers

Membrane Trafficking of the Cystic Fibrosis Gene Product, Cystic Fibrosis Transmembrane Conductance Regulator, Tagged with Green Fluorescent Protein in Madin-Darby Canine Kidney Cells

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