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Mountain View Acres, CA, United States

Afshari A.,California State University, East Bay | Uhde-Stone C.,California State University, East Bay | Lu B.,System Biosciences SBI
Biochemical and Biophysical Research Communications | Year: 2014

Despite their fundamental importance, the dynamics of signaling pathways in living cells remain challenging to study, due to a lack of non-invasive tools for temporal assessment of signal transduction in desired cell models. Here we report a dual-reporter strategy that enables researchers to monitor signal transduction in mammalian cells in real-time, both temporally and quantitatively. This is achieved by co-expressing green fluorescent protein and firefly luciferase in response to signaling stimuli. To display the versatility of this approach, we constructed and assessed eight unique signaling pathway reporters. We further validated the system by establishing stable NF-κB pathway reporter cell lines. Using these stable cell lines, we monitored the activity of NF-κB-mediated inflammatory pathway in real-time, both visually and quantitatively. Live visualization has the power to reveal individual cell responses and is compatible with single cell analysis, In addition, we provide evidence that this system is readily amenable to a high-throughput format. Together, our findings demonstrate the potential of the dual reporter system, which significantly improves the capacity to study signal transduction pathways in mammalian cells. © 2014 Elsevier Inc. All rights reserved. Source


Afshari A.,California State University, East Bay | Uhde-Stone C.,California State University, East Bay | Lu B.,System Biosciences SBI
Biochemical and Biophysical Research Communications | Year: 2015

Luciferase assay has become an increasingly important technique to monitor a wide range of biological processes. However, the mainstay protocols require a luminometer to acquire and process the data, therefore limiting its application to specialized research labs.To overcome this limitation, we have developed an alternative protocol that utilizes a commonly available cooled charge-coupled device (CCCD), instead of a luminometer for data acquiring and processing. By measuring activities of different luciferases, we characterized their substrate specificity, assay linearity, signal-to-noise levels, and fold-changes via CCCD. Next, we defined the assay parameters that are critical for appropriate use of CCCD for different luciferases. To demonstrate the usefulness in cultured mammalian cells, we conducted a case study to examine NFκB gene activation in response to inflammatory signals in human embryonic kidney cells (HEK293 cells). We found that data collected by CCCD camera was equivalent to those acquired by luminometer, thus validating the assay protocol. In comparison, The CCCD-based protocol is readily amenable to live-cell and high-throughput applications, offering fast simultaneous data acquisition and visual and quantitative data presentation.In conclusion, the CCCD-based protocol provides a useful alternative for monitoring luciferase reporters. The wide availability of CCCD will enable more researchers to use luciferases to monitor and quantify biological processes. © 2015 Elsevier Inc. Source


Uhde-Stone C.,California State University, East Bay | Cheung E.,System Biosciences SBI | Lu B.,System Biosciences SBI
Biochemical and Biophysical Research Communications | Year: 2014

Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells. © 2013 Elsevier Inc. All rights reserved. Source


Uhde-Stone C.,California State University, East Bay | Sarkar N.,System Biosciences SBI | Antes T.,System Biosciences SBI | Otoc N.,System Biosciences SBI | And 3 more authors.
RNA | Year: 2014

Significant progress in the functional understanding of microRNAs (miRNAs) has been made in mice, but a need remains to develop efficient tools for bi-allelic knockouts of miRNA in the human genome. Transcription activator-like effector nucleases (TALENs) provide an exciting platform for targeted gene ablation in cultured human cells, but bi-allelic modifications induced by TALENs alone occur at low frequency, making screening for double knockouts a tedious task. Here, we present an approach that is highly efficient in bi-allelic miRNA ablation in the human genome by combining TALENs targeting to the miRNA seed region with a homologous recombination donor vector and a positive selection strategy. A pilot test of this approach demonstrates bi-allelic miR-21 gene disruption at high frequency (∼87%) in cultured HEK293 cells. Analysis of three independent clones showed a total loss of miR-21 expression. Phenotypical analysis revealed increased miR-21 target gene expression, reduced cell proliferation, and alterations of global miRNA expression profiles. Taken together, our study reveals a feasible and efficient approach for biallelic miRNA ablation in cultured human cells and demonstrates its usefulness in elucidating miRNA function in human cells. © 2014 Uhde-Stone et al. Source


Uhde-Stone C.,California State University, East Bay | Huang J.,System Biosciences SBI | Lu B.,System Biosciences SBI
Biological Procedures Online | Year: 2012

Background: Transcription activator-like effectors (TALEs) are a class of naturally occurring transcription effectors that recognize specific DNA sequences and modulate gene expression. The modularity of TALEs DNA binding domain enables sequence-specific perturbation and offers broad applications in genetic and epigenetic studies. Although the efficient construction of TALEs has been established, robust functional tools to assess their functions remain lacking. Results: We established a dual reporter system that was specifically designed for real-time monitoring and quantifying gene expression mediated by TALEs. We validated both sensitivity and specificity of this dual-reporter system in mammalian cells, and demonstrated that this dual reporter system is robust and potentially amenable to high throughput (HTP) applications. Conclusion: We have designed, constructed and validated a novel dual reporter system for assessing TALE mediated gene regulations. This system offers a robust and easy-to- use tool for real-time monitoring and quantifying gene expression in mammalian cells. © 2012 Uhde-Stone et al.; licensee BioMed Central Ltd. Source

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