Impacts of different promoters on the mammalian one-hybrid assay for detecting nuclear receptor agonists

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Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
11:02

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Published on: May 27, 2016

Zhi-Hui Zheng1, Xin-Hua Lu, Hua Zhang

  • 1College of Life Science, Hebei Normal University, Shijiazhuang, 050016, China.

Analytical and Bioanalytical Chemistry
|January 19, 2010
PubMed

Insights

Choosing the right promoter is crucial for nuclear receptor drug discovery assays. The SV40 promoter enhances high-throughput screening (HTS) assays for detecting nuclear receptor ligands, offering improved sensitivity and reproducibility over TATA box promoters.

Area of Science:

  • Molecular Biology
  • Drug Discovery
  • Biochemistry

Background:

  • Nuclear receptors are critical transcription factors and key targets in drug discovery.
  • Mammalian one-hybrid systems enable cell-based high-throughput screening (HTS) for nuclear receptor ligands.

Purpose of the Study:

  • To investigate the impact of different promoters on the performance of nuclear receptor HTS assays.
  • To identify an optimal promoter for robust and sensitive detection of nuclear receptor ligands.

Main Methods:

  • Development and comparison of cell-based HTS assays using reporter vectors with different promoters (SV40 vs. TATA box).
  • Evaluation of assay performance based on signal/noise ratios, Z' factors, coefficient variation, sensitivity, and hit rates.

Main Results:

  • The SV40 promoter significantly improved HTS assay performance, yielding higher signal/noise ratios, acceptable Z' factors (>0.6), and low coefficient variation (<12.5%).
  • Assays utilizing the SV40 promoter demonstrated increased robustness, reproducibility, sensitivity, and a higher hit rate compared to TATA box promoters.
  • The SV40 promoter facilitated longer signal decay times and easier miniaturization for 384-well formats.

Conclusions:

  • Promoter selection is a critical determinant in developing effective reporter gene HTS assays for nuclear receptors.
  • The SV40 promoter is more adaptable and superior to the TATA box minimal promoter for Mammalian one-hybrid HTS assays detecting nuclear receptor agonists.

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