Understanding, improving and using green fluorescent proteins

Cubitt, A B; Heim, Roger; Adams, Stephen; Boyd, Aileen E.; Gross, Larry A.; Tsien, Roger Y.

doi:10.1016/s0968-0004(00)89099-4

Cited by 1,271 publications

(984 citation statements)

References 35 publications

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“…In this paper all cells transiently transfected with GFP under anoxic conditions showed a reduction in highly fluorescent cells (thousand-fold above autofluorescence) when compared to those transfected in air or mild hypoxia. It is known that the marker protein GFP requires molecular oxygen to fold correctly and fluoresce (Cubitt et al, 1995). Analysis of stable GFP transfectants had shown that anoxia reduced GFP fluorescence, whereas less severe hypoxia (including gassing with 95% N 2 , 5% CO 2 ) did not significantly alter GFP fluorescence (Coralli et al, manuscript in preparation).…”

Section: Discussionmentioning

confidence: 99%

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Gene delivery to hypoxic cells in vitro

et al. 2000

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Summary Hypoxia in solid tumours has been correlated with poor prognosis and resistance to radiation and chemotherapy. Hypoxia is also a strong stimulus for gene expression. We previously proposed a gene therapy approach which exploits the presence of severe hypoxia in tumours for the induction of therapeutic genes. Hypoxic cells are known to have a reduced metabolic rate, transcription and translation. These facts may prevent gene transfer and therefore warranted further investigation. In this paper the feasibility of gene delivery in vitro under tumour conditions was demonstrated. DNA was delivered in vitro using a peptide-mediated non-viral system. Across a range of oxygen tensions and mammalian cell lines (including human tumour and endothelial cells) it was shown that hypoxic cells could be transfected. Transfection efficiencies varied depending on the level of hypoxia, cell characteristics and gene promoters used. An in vitro model of hypoxia/reoxygenation, designed to mimic the variable nature of tumour hypoxia, showed that hypoxic preconditioning and reoxygenation alone did not reduce transfection efficiency significantly; only chronic anoxia reduced transfection. The fact that neither intermediate hypoxia nor intermittent anoxia significantly reduced transfection is promising for future hypoxia-targeted gene therapy strategies. 662-667 © 2000 Cancer Research Campaign doi: 10.1054/ bjoc.2000.1318, available online at http://www.idealibrary.com on List of abbreviations: HIF-1, hypoxia inducible factor 1; CMV, cytomegalovirus; SV40, simian virus 40; AnO 2 , anoxia; GFP, green fluorescent protein; FACS, fluorescent activated cell sorting Transfection efficiencies of two commonly used marker genes and viral promoters were recorded in a range of cell lines under different oxygenation conditions. A simple in vitro model of hypoxia/reoxygenation was used to mimic the variable nature of tumour hypoxia. MATERIALS AND METHODS Cell lines and growth mediaThe human bladder carcinoma cell line T24 (European Collection of Cell Cultures; Bubenik et al, 1973;Dirks et al, 1999), the immortalized human microvascular endothelial cell line HMEC-1 (Ades et al, 1992), human squamous carcinoma cell line of the head and neck FaDu (Rangan, 1972; American Type Culture Collection), the mouse hepatoma cell line Hepa-1 wild type (clone Hepa-1c1c7) and its derivative Hepa c4 (HIF-1β mutant; Hankinson, 1979) were utilised. Only cells tested negative for mycoplasma were used in the experiments, since it had been shown that mycoplasma interfered with transfection, especially under hypoxia (results not shown). All cells were maintained in Dulbecco's MEM (DMEM, Life Technologies, UK) with 10% fetal calf serum and L-glutamine in a 5% CO 2 humidified incubator at 37°C.Cell proliferation was monitored by cell counting using a haemocytometer and trypan blue (0.2% final concentration, Sigma) to distinguish live and dead cells. Hypoxic conditionsCells were plated 16-24 h prior to the experiment in oxygen impermeable dishes (Permanox, Nunc, UK...

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Section: Discussionmentioning

confidence: 99%

“…The GFP chromophore is formed by a cyclization reaction and an oxidation step that requires molecular oxygen (Cubitt et al, 1995). It is also known that some gene promoters are regulated by oxygenation.…”

Section: Hypoxic Transfection Efficiency: Effect Of Gene Product and mentioning

confidence: 99%

Gene delivery to hypoxic cells in vitro

et al. 2000

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“…33 Therefore, only an empirical analysis of VP22 fusion proteins is able to elucidate which fusion products maintain the functionality of both components.…”

Section: Mh3924a Cells Seeded In 24-well Plates Were Transduced With mentioning

confidence: 99%

Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene

et al. 2001

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“…Derivatives of the green fluorescent protein cloned from Aequora victoria have been used extensively in biological research based on the assumption that it does not significantly alter the function of its fusion partner [37,38]. Yet there are occasional reports of functional changes arising in proteins as a consequence of the relative placement of the GFP fusion partner [39,40].…”

Section: Discussionmentioning

confidence: 99%

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

Lee¹,

Duhé²

2006

J Biomed Sci

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SummaryJanus kinase 2 (JAK2) is an essential intracellular signal transducer for numerous cytokines and hormones. To examine how JAK2 structural modifications can affect cellular physiology, we created expression vectors for chimeric proteins containing an enhanced green fluorescent protein (EGFP) fused to rat JAK2 (EGFP/rJAK2), and a kinase-inactive variant, EGFP/rJAK2(K882E). The properties of EGFP/rJAK2 were examined following transient transfection of COS-7 cells. EGFP/rJAK2 was expressed throughout the cell, and was found in subcellular membrane, cytosolic and nuclear fractions. Interestingly, EGFP/rJAK2 phosphorylated other proteins in situ without additional cytokine stimulation. Furthermore, despite a much higher level of tyrosine phosphorylation arising from in situ autophosphorylation, the in vitro radiolabelling autokinase activity of EGFP/rJAK2 was significantly less than that of the endogenous JAK2. These results reveal a technical limitation of the application of the ''conventional'' in vitro radiolabelling autokinase assay to hyperphosphorylated forms of the enzyme and illustrate the potential weaknesses in individual assays commonly used to determine JAK2's enzymatic activity and subcellular distribution. We also suggest that the EGFP/rJAK2 model can be very useful in studying JAK2-related cancers, because its ubiquitous distribution and abnormal constitutive hyperphosphorylation may distinguish it from the cytokine-regulated, membrane-proximal form of JAK2 associated with normal physiology.

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Understanding, improving and using green fluorescent proteins

Cited by 1,271 publications

References 35 publications

Gene delivery to hypoxic cells in vitro

Gene delivery to hypoxic cells in vitro

Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

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