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IMAGEtag (Intracellular MultiAptamer Genetic tag) for Real‐time Imaging of Gene Promoter Activity
Author(s) -
Shin Ilchung,
Ilgu Muslum,
Haarberg H. Eirik,
Gupta Vinayak,
Kraus George A,
NilsenHamilton Marit
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.903.2
Subject(s) - aptamer , förster resonance energy transfer , reporter gene , promoter , green fluorescent protein , rna , transcription (linguistics) , gene , biology , intracellular , luciferase , microbiology and biotechnology , yeast , computational biology , gene expression , chemistry , fluorescence , biochemistry , transfection , physics , linguistics , philosophy , quantum mechanics
Reporter proteins such as fluorescence proteins and luciferase allow for non‐invasive detection of the products of gene expression in living cells. However, current reporter proteins do not provide for real‐time imaging of promoter activity in living cells. This is because of the long time after transcription required for RNA maturation, protein synthesis and maturation. We have developed an alternative reporter system for imaging real‐time using strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer Genetic tags), which can be expressed from a promoter of choice. The tobramycin and neomycin RNA aptamers have so far been utilized for this system and expressed in yeast from the GAL1 promoter. For imaging, the cells are incubated with their ligands that are labeled with fluorescent dyes. To increase signal/noise, ligands have been separately conjugated with the FRET (Förster resonance energy transfer) pairs, Cy3 and Cy5. With these constructs, the transcribed aptamers can be imaged after induction by galactose. Initial optimization of IMAGEtags for imaging is done with yeast followed by the application of successful IMAGEtags to detect rapid changes in transcriptional activity in animal and plant cells in culture. This work was supported by the National Institutes of Health and the Biological Systems Science Division, Office of Energy Science Research of the U. S. Department of Energy.