Green Fluorescent Protein as a Marker for Gene Expression

Chalfie, Martin; Tu, Yuan; Euskirchen, Ghia; Ward, William W.; Prasher, Douglas C.

doi:10.1126/science.8303295

Cited by 5,988 publications

(2,123 citation statements)

References 33 publications

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“…In particular, it was recently shown that PTEN negatively regulates cell survival conferring enhanced sensitivity to di erent apoptotic stimuli . To discriminate between these possibilities we made use of a PTEN-encoding construct in which the full-length cDNA coding for PTEN was fused in frame with the auto¯uorescent Eukaryotic Green Fluorescent Protein (EGFP) or with the N-terminal FLAG tag (Chal®e et al, 1994). All experiments described below were performed three times in duplicate in which at least 200 transfected cells were counted.…”

Section: Constitutive Pten Expression Inhibits S Phase Entry In Thyromentioning

confidence: 99%

PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells is mediated by p27kip1

Bruni¹,

Boccia²,

Baldassarre³

et al. 2000

Oncogene

138

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The dual-speci®city phosphatase PTEN/MMAC1/TEP1 has recently been identi®ed as the tumor suppressor gene most frequently mutated and/or deleted in human tumors. Germline mutations of PTEN give rise to Cowden Disease (CD), an autosomal dominantlyinherited cancer syndrome which predisposes to increased risk of developing breast and thyroid tumors. However, PTEN mutations have rarely been detected in sporadic thyroid carcinomas. In this study, we con®rm that PTEN mutations in sporadic thyroid cancer are infrequent as we found one point mutation and one heterozygous deletion of PTEN gene in 26 tumors and eight cell lines screened. However, we report that PTEN expression is reduced ± both at the mRNA and at the protein level ± in ®ve out of eight tumor-derived cell lines and in 24 out of 61 primary tumors. In most cases, decreased PTEN expression is correlated with increased phosphorylation of the PTEN-regulated protein kinase Akt/PKB. Moreover, we demonstrate that PTEN may act as a suppressor of thyroid cancerogenesis as the constitutive re-expression of PTEN into two di erent thyroid tumor cell lines markedly inhibits cell growth. PTEN-dependent inhibition of BrdU incorporation is accompanied by enhanced expression of the cyclin-dependent kinase inhibitor p27 kip1 and can be overcome by simultaneous co-transfection of an excess p27 kip1 antisense plasmid. Accordingly, in a subset of thyroid primary carcinomas and tumor-derived cell lines, a striking correlation between PTEN expression and the level of p27 kip1 protein was observed. In conclusion, our ®ndings demonstrate that inactivation of PTEN may play a role in the development of sporadic thyroid carcinomas and that one key target of PTEN suppressor activity is represented by the cyclin-dependent kinase inhibitor p27 kip1 . Oncogene (2000) 19, 3146 ± 3155.

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Section: Constitutive Pten Expression Inhibits S Phase Entry In Thyromentioning

confidence: 99%

PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells is mediated by p27kip1

Bruni¹,

Boccia²,

Baldassarre³

et al. 2000

Oncogene

138

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“…The invention of fluorescent proteins (FPs) as genetically encoded reporters in the early nineties revolutionized the cell biologist's ability to gain insight into cellular processes in living cells. Since the first application of the original Aequorea Victoria wild type green fluorescent protein (GFP) in nematodes [1] researchers started to generate improved FP versions which have better photophysical properties and optimal behavior in cells and organisms [2][3]. Nowadays, the scientific community has access to a vast number of FPs which cover a broad spectral range and have become an indispensable tool to study cell biology by fluorescence microscopy.…”

Section: Introductionmentioning

confidence: 99%

Chemical tags: Applications in live cell fluorescence imaging

Wombacher

Cornish

2011

Journal of Biophotonics

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Technologies to visualize cellular structures and dynamics enable cell biologists to gain insight into complex biological processes. Currently, fluorescent proteins are used routinely to investigate the behavior of proteins in live cells. Chemical biology techniques for selective labeling of proteins with fluorescent labels have become an attractive alternative to fluorescent protein labeling. In the last ten years the progress in the development of chemical tagging methods have been substantial offering a broad palette of applications for live cell fluorescent microscopy. Several methods for protein labeling have been established, using protein tags, peptide tags and enzyme mediated tagging. This review focuses on the different strategies to achieve the attachment of fluorophores to proteins in live cells and cast light on the advantages and disadvantages of each individual method. Selected experiments in which chemical tags have been successfully applied to live cell imaging will be discussed and evaluated. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

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“…An unpaired two‐tailed Student's t ‐test was used to evaluate the gene expression difference in data from two different groups, including the following: before and after chemical treatments (the gene profiles of chemical treatment vs. those incubated with dimethyl sulfoxide (DMSO) control) in MCF7, PC3, and A375 cells at 6 hours and 24 hours, respectively; before and after shRNAs/overexpression perturbations (the gene knockdown/overexpression group vs. its different control groups, such as empty vector GFP,18 eGFP,19 Luciferase, HcRed,20 or lacZ,21 respectively). The difference was considered significant if P < 0.01.…”

Section: Methodsmentioning

confidence: 99%

Systematic Quality Control Analysis of LINCS Data

Cheng

2016

CPT Pharmacom & Syst Pharma

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The Library of Integrated Cellular Signatures (LINCS) project provides comprehensive transcriptome profiling of human cell lines before and after chemical and genetic perturbations. Its L1000 platform utilizes 978 landmark genes to infer the transcript levels of 14,292 genes computationally. Here we conducted the L1000 data quality control analysis by using MCF7, PC3, and A375 cell lines as representative examples. Before perturbations, a promising 80% correlation in transcriptome was observed between L1000‐ and Affymetrix HU133A‐platforms. After library‐based shRNA perturbations, a moderate 30% of differentially expressed genes overlapped between any two selected controls viral vectors using the L1000 platform. The mitogen‐activated protein kinase, vascular endothelial growth factor, and T‐cell receptor pathways were identified as the most significantly shared pathways between chemical and genetic perturbations in cancer cells. In conclusion, L1000 platform is reliable in assessing transcriptome before perturbation. Its response to perturbagens needs to be interpreted with caution. A quality control analysis pipeline of L1000 is recommended before addressing biological questions.

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Green Fluorescent Protein as a Marker for Gene Expression

Cited by 5,988 publications

References 33 publications

PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells is mediated by p27kip1

PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells is mediated by p27kip1

Chemical tags: Applications in live cell fluorescence imaging

Systematic Quality Control Analysis of LINCS Data

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