Cellular Ca2+-Responding Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-palindromic NFAT-Response Elements

Methods Mol Biol. 2019:1929:95-109. doi: 10.1007/978-1-4939-9030-6_7.

Abstract

Luciferase reporter gene systems based on the NFAT-response element (RE) have been used to monitor intracellular Ca2+ elevation. However, Ca2+ mobilization agent (e.g., ionomycin) alone is not adequate to activate the currently often employed reporter gene that contains the NFAT-RE found in the IL2 promoter. In addition to activation of NFAT through the Ca2+-calmodulin/calcineurin pathway, activation of AP-1 as a partner transcription factor is essential for the IL2-based NFAT-RE system. Here, we describe a detailed method for the recently developed new reporter gene system containing the NFAT-RE from the IL8 promoter. This system enables us to monitor endpoint effects of Ca2+-mobilizing agonists independent of AP-1 activation.

Keywords: Calcineurin; Calcium signaling; Calmodulin; Carbachol; Luminescence; NFAT; NanoLuc; Reporter gene assay; Transcription factor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calcineurin / genetics
  • Calcium / analysis*
  • Calmodulin / genetics
  • Gene Expression Regulation
  • Genes, Reporter*
  • HEK293 Cells
  • Humans
  • Interleukin-2 / chemistry
  • Interleukin-2 / genetics*
  • NFATC Transcription Factors / metabolism
  • Promoter Regions, Genetic
  • Transcription Factor AP-1 / genetics
  • Transcriptional Activation

Substances

  • Calmodulin
  • IL2 protein, human
  • Interleukin-2
  • NFATC Transcription Factors
  • Transcription Factor AP-1
  • Calcineurin
  • Calcium