Establishment of Tetracycline-Inducible, Survivin-Expressing CHO Cell Lines by an Optimized Screening Method

Zhao, Xinghui; Yu, Yanping; Zhao, Zhongchao; Guo, Jia; Fu, Ling; Takata, Yasuyuki; Hou, Lihua; Yi, Shaoqiong; Chen, Wei

doi:10.1271/bbb.120395

Cited by 4 publications

(5 citation statements)

References 12 publications

(9 reference statements)

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“…Besides, the fluorescent intensity of fluorescent proteins can be associated with its protein expression level [22–24]. Therefore, if the fluorescent proteins are overly stable, there is a limitation for such reporter system to analyze some precise regulations of target gene.…”

Section: Resultsmentioning

confidence: 99%

“…Although chemiluminescence is more sensitive than fluorescence in nearly all systems, fluorescent reporters still have some advantages; for example, fluorescent proteins were suited to bioimaging assays in living cells which does not require cell permeabilization or the addition of exogenous substrates [ 18 ]. Besides, the fluorescent intensity of fluorescent proteins can be associated with its protein expression level [ 22 – 24 ]. Therefore, if the fluorescent proteins are overly stable, there is a limitation for such reporter system to analyze some precise regulations of target gene.…”

Section: Resultsmentioning

confidence: 99%

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Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System

Yang

Kuo

Chuang

et al. 2013

BioMed Research International

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The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB) is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP) at C-terminus and red fluorescent protein (RFP, DsRed) at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System

Yang

Kuo

Chuang

et al. 2013

BioMed Research International

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“…Therefore, it is not a surprise Fluorescent reporters are well suited for bio-imaging assays in living cells that do not require cell permeabilization or the addition of exogenous substrates [28]. Moreover, the fluorescence intensity of the fluorescent proteins can be correlated with the protein expression level [29,30]. Therefore, we used the stable cells in combination with the green fluorescent reporter as a rapid and realistic assay system to analyze the regulation of melanogenesis.…”

Section: Construction Of the Mewo/pmitf-egfp Mewo/ptyr-egfp And Mewomentioning

confidence: 99%

Establishment of a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the melanogenesis-related genes in human melanoma cells

Lin

Yang

Lin

et al. 2015

Enzyme and Microbial Technology

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“… 18 In both cases, the VP16 domain recruits RNA polymerase (RNAP) when TetR is bound to a synthetic, TRE-containing promoter. These switches typically have low basal expression, exhibit a large dynamic range (from 10 to several thousand-fold induction), and have been shown to function in a wide range of tissue culture systems, including embryonic stem cells, 19 , 20 CHO, 21 HEK, 22 HeLa, 23 and MCF-7 24 cells, as well as in living animals. 25 …”

mentioning

confidence: 99%

“…18 In both cases, the VP16 domain recruits RNA polymerase (RNAP) when TetR is bound to a synthetic, TREcontaining promoter. These switches typically have low basal expression, exhibit a large dynamic range (from 10 to several thousand-fold induction), and have been shown to function in a wide range of tissue culture systems, including embryonic stem cells, 19,20 CHO, 21 HEK, 22 HeLa, 23 and MCF-7 24 cells, as well as in living animals. 25 Homologues of TetR have been used to build synthetic gene switches for various applications, 2,26 and switches responding to other antibiotics, including erythromycin (MphR) 27 and pristinamycin (Pip) 28 have also been constructed.…”

mentioning

confidence: 99%

Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

et al. 2014

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Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >105 members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 μM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.

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Establishment of Tetracycline-Inducible, Survivin-Expressing CHO Cell Lines by an Optimized Screening Method

Cited by 4 publications

References 12 publications

Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System

Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System

Establishment of a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the melanogenesis-related genes in human melanoma cells

Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

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