In vitro MR imaging of regulated gene expression.
Ключевые слова
абстрактный
OBJECTIVE
To design and evaluate a construct that allows regulated expression of the magnetic resonance (MR) imaging reporter gene human tyrosinase under control of the tetracycline response element.
METHODS
A breast cancer cell line (MCF-7) was transfected with a plasmid that codes for the tetracycline-controlled transactivator and a new construct. In this construct, the reporter gene human tyrosinase is under control of the tetracycline response element, thus allowing suppression of gene expression by adding doxycycline (tetracycline switched off). A reverse transcription polymerase chain reaction was conducted to evaluate gene expression. Additionally, immunohistochemical investigation of tyrosinase and melanin staining was undertaken to analyze the presence of these molecules. After culture in an iron- and holotransferrin-enriched medium, cells were imaged in a 1.0-T clinical MR imager by using a surface coil and T1-weighted spin-echo and gradient-echo sequences.
RESULTS
Two stable transfected cell clones were established. Cells cultured with doxycycline showed no background expression of the human tyrosinase gene, whereas withdrawal of doxycycline resulted in detectable tyrosinase messenger RNA expression. Gene expression results in a detectable tyrosinase protein level and melanin content. Increased signal intensity on T1-weighted MR images in cells that expressed the reporter gene was observed in comparison to genetically identical cells with the reporter gene switched off.
CONCLUSIONS
Our construct enables MR imaging of regulated tyrosinase gene expression in vitro.
