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A Reporter Based Cellular Assay for Monitoring Splicing Efficiency
Published on: September 15, 2021
Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various
Hitoshi Shiku1, Michiaki Takeda, Tatsuya Murata
1Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579, Japan. shiku@bioinfo.che.tohoku.ac.jp
Analytica Chimica Acta
|April 14, 2009
Summary
Scanning electrochemical microscopy (SECM) offers a sensitive method for detecting cellular responses. This electrochemical assay enables parallel evaluation of multiple HeLa cell lines, significantly reducing cell count requirements compared to traditional methods.
Area of Science:
- Biomedical Engineering
- Cell Biology
- Analytical Chemistry
Background:
- Reporter gene assays are crucial for studying cellular signaling pathways.
- Existing methods often require large cell numbers and lack parallel evaluation capabilities.
- Scanning electrochemical microscopy (SECM) presents a potential alternative for sensitive, high-throughput analysis.
Purpose of the Study:
- To develop and validate an SECM-based electrochemical assay for analyzing HeLa cell responses.
- To assess the sensitivity and efficiency of SECM compared to conventional chemiluminescence (CL) assays.
- To enable parallel evaluation of different reporter gene constructs in a single experiment.
Main Methods:
- HeLa cells were transfected with plasmid vectors encoding secreted alkaline phosphatase (SEAP) under the control of various response elements (GRE, CRE, kappaB).
- Cells were patterned into a 4x4 array using poly(dimethylsiloxane) (PDMS) stencils and exposed to specific stimuli (dexamethasone, forskolin, TNF-alpha).
- Electrochemical measurements were performed using SECM to detect p-aminophenol (PAP) generated from p-aminophenylphosphate (PAPP) hydrolysis.
Main Results:
- SECM successfully detected the enzymatic activity of SEAP in response to specific stimuli in transfected HeLa cells.
- The assay allowed for parallel evaluation of pGRE-SEAP, pCRE-SEAP, and pNFkappaB-SEAP constructs on the same chip.
- The SECM method demonstrated high sensitivity, requiring only 300 cells per pattern, a significant reduction from the 5x10^4 cells needed for CL assays.
Conclusions:
- SECM provides a highly sensitive and efficient platform for analyzing cellular responses mediated by reporter genes.
- This electrochemical approach enables parallel screening of multiple signaling pathways with reduced cell input.
- SECM offers a promising alternative to conventional methods for drug screening and toxicological studies.
