A novel reporter system for cyclic AMP mediated gene expression in mammalian cells based on synthetic transgene expression system.

Affiliation

Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, India. Electronic address: [Email]

Abstract

Cyclic AMP (cAMP) is an important second messenger that mediates various biological functions in both prokaryotes and eukaryotes. Due to the ever increasing significance in studying the function and modulation of cAMP-based signaling, it is important to develop a protein-based biosensor that reports the cAMP mediated gene expression. Based on a synthetic transgene approach, an artificial mammalian transactivator was developed by fusing a transcriptional regulatory element cAMP receptor protein (CRP) of Escherichia coli to the VP16 transactivation domain of Herpes simplex virus in a mammalian expression vector (pLA1) that activates CRP specific operator site present in a chimeric promoter (OCRP- PhCMVmin- Luciferase) in a concentration dependent manner in mammalian cells. Our results reveal that the engineered transactivator report the gene expression mediated by cAMP directly in mammalian cells and this cAMP reporter system works irrespective of Protein kinase A (PKA) - cyclic AMP response element binding protein (CREB) - cyclic AMP response element (CRE) signaling since the luciferase activity mediated by synthetic gene construct is seen even in the presence of PKA inhibitor H-89 (derived from H-8 (N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide). Furthermore this synthetic transcription factor plays a significant role in reporting and mediating cAMP signaling in tumorigenic cells which possess an aberrant cAMP signaling due to PKA and CREB mutations.

Keywords

CRP,Mammalian cAMP transactivator,Synthetic biology,Synthetic gene circuit,cAMP,

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