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      Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter

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          Abstract

          Constitutive promoters are used routinely to drive ectopic gene expression. Here, we carried out a systematic comparison of eight commonly used constitutive promoters (SV40, CMV, UBC, EF1A, PGK and CAGG for mammalian systems, and COPIA and ACT5C for Drosophila systems). We also included in the comparison the TRE promoter, which can be activated by the rtTA transcriptional activator in a doxycycline-inducible manner. To make our findings representative, we conducted the comparison in a variety of cell types derived from several species. We found that these promoters vary considerably from one another in their strength. Most promoters have fairly consistent strengths across different cell types, but the CMV promoter can vary considerably from cell type to cell type. At maximal induction, the TRE promoter is comparable to a strong constitutive promoter. These results should facilitate more rational choices of promoters in ectopic gene expression studies.

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          Most cited references16

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          Cell lines derived from late embryonic stages of Drosophila melanogaster.

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            Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor.

            The neurogenic loci Notch and Delta, which both encode EGF-homologous transmembrane proteins, appear to function together in mediating cell-cell communication and have been shown to interact at the cell surface in vitro. To examine the role of the EGF repeats in this interaction, we performed an extensive deletion mutagenesis of the extracellular domain of Notch. We find that of the 36 EGF repeats of Notch, only two, 11 and 12, are both necessary and sufficient to mediate interactions with Delta. Furthermore, this Delta binding ability is conserved in the corresponding two repeats from the Xenopus Notch homolog. We report a novel molecular interaction between Notch and Serrate, another EGF-homologous transmembrane protein containing a region of striking similarity to Delta, and show that the same two EGF repeats of Notch also constitute a Serrate binding domain. These results suggest that Notch may act as a multifunctional receptor whose 36 EGF repeats form a tandem array of discrete ligand-binding units, each of which may potentially interact with several different proteins during development.
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              Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle.

              Although the transient nature of transgene expression using first-generation adenovirus (Ad) vectors is well known, the exact mechanisms responsible for this phenomenon are uncertain. Rats were given intramuscular (i.m.) injections of a first-generation Ad containing the human fibroblast growth factor 4 (hFGF-4) gene driven by the cytomegalovirus (CMV) promoter and enhancer (CMV-PE). The copy number of hFGF-4 mRNA and viral DNA was measured in the same muscles by quantitative RT-PCR and quantitative PCR at times between 1 h and 84 days after virus injection. Quantitative Southern blot analysis for the intact hFGF-4 transcription unit DNA was also performed, and the methylation status of the CMV-PE DNA in the muscle was determined using bisulfite sequencing. The copy number of hFGF-4 mRNA peaked at 6 h then decreased 56-fold by 24 h, and a further 240-fold between days 3 and 28. Although the viral DNA copy number also decreased 23-fold between 6 h and 28 days, the ratio of copies of hFGF-4 mRNA per copy of viral DNA decreased 385-fold over this period. Methylation of the CMV-PE DNA in the muscle at both CpG and non-CpG sites was observed 24 h after virus administration and had increased at day 7. Decreased transcription associated with extensive methylation of the CMV-PE was the major mechanism responsible for the decrease in transgene mRNA levels. Strategies for preventing transcriptional silencing will be valuable for improving the duration of transgene expression from adenoviral vectors. Copyright 2004 John Wiley & Sons, Ltd.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2010
                12 May 2010
                : 5
                : 5
                : e10611
                Affiliations
                [1 ]School of Life Sciences, Northeast Normal University, Changchun, China
                [2 ]Howard Hughes Medical Institute, Department of Human Genetics, University of Chicago, Chicago, Illinois, United States of America
                [3 ]Center for Stem Cell Biology and Tissue Engineering, Sun Yat-sen University, The Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Guangzhou, China
                New Mexico State University, United States of America
                Author notes

                Conceived and designed the experiments: APX BTL. Performed the experiments: JYQ LZ. Analyzed the data: JYQ BTL. Contributed reagents/materials/analysis tools: LZ KC IH APX. Wrote the paper: JYQ BTL. Supervised JYQ: BZR.

                Article
                10-PONE-RA-16503R1
                10.1371/journal.pone.0010611
                2868906
                20485554
                2538cb46-baa0-479f-a93e-662fc440aefd
                Qin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 22 February 2010
                : 16 April 2010
                Page count
                Pages: 4
                Categories
                Research Article
                Molecular Biology
                Cell Biology/Gene Expression
                Genetics and Genomics/Gene Expression

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