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IMAGEtags for imaging gene expression in living cells in real‐time
Author(s) -
Ilgu Muslum,
Shin Ilchung,
Haarberg Hans Eirik,
Gupta Vinayak,
Kraus George A,
NilsenHamilton Marit
Publication year - 2009
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.23.1_supplement.517.2
Subject(s) - reporter gene , aptamer , green fluorescent protein , microbiology and biotechnology , luciferase , fluorescence lifetime imaging microscopy , gene expression , molecular imaging , preclinical imaging , live cell imaging , in vivo , biology , computational biology , gene , cell , transfection , fluorescence , biochemistry , genetics , physics , quantum mechanics
The use of green fluorescent protein (GFP) and luciferase in cellular imaging has enabled significant advances in cellular imaging in vitro and in vivo . However, these imaging systems have the disadvantages of very slow response times and a high energy costs for synthesizing and maintaining reporter protein levels. For example, they are not amendable to imaging changes in gene expression in real time. Also, the energy requirements of protein reporter systems are potentially debilitating in vivo when cells such as stem cells need to proliferate and populate a niche in a damaged tissue where the oxygen and nutrient concentrations are limiting. Therefore, our goal is to establish an alternative reporter system that can be used for real‐time imaging and for imaging stem cells in vivo with minimal metabolic perturbation. This new reporter system involves the use of IMAGEtags ( I ntracellular M ultiaptamer Ge netic tags) , which are strings of RNA aptamers expressed from a promoter of choice. To establish this RNA‐based reporter system, we utilized available aptamers that recognize small target molecules such as neomycin, tobramycin and theophylline and constructed vectors that express multiple repeats of these aptamers. The target molecules were labeled with fluorescent moieties for intracellular imaging. To increase signal/noise the aptamer targets were separately labeled with fluorescent FRET pairs. IMAGEtags are currently being developed for reporting gene expression changes in yeast and cultured mammalian cells. Once optimized for imaging, this new reporter system will have imaging applications such as real‐time detection of changes in gene expression and mRNA processing, and tracking cell movements in vivo . Supported by grants from the NIH (R01HL078659) and the DOE (OBER).