CREATION OF U251 SENSING CELL LINES THROUGH LIPOFECTION WITH NUCLEUS AND CYTOPLASM HAVING DIFFERENT OPTICAL SIGNATURES

Antonio Adami Pires*, Brian Strauss and Eugenia Costanzi-Strauss

ABSTRACT

The bioactivity of new drugs may result in morphological alterations at the single cell level that are indicative of cell vitality. With current technology permits the detection of perturbations in the cell cycle, such as mitosis, senescence or death, using non-invasive methods in live cells. Our objective is to develop sensor lines for the real time visualization of changes in chromatin or cytoplasmic morphology. A palate of optical markers such as fusion proteins that contain histone H2B and a fluorescent protein, such as Katushka would reveal nuclear alterations. Changes in the cytoplasm can be revealed using a fusion between actin and CFP. For example, a cell entering senescence could be identified by distinct optical signatures such as weakly stained, relaxed chromatin and an enlarged, flattened cytoplasm. A cell in mitosis would be characterized by condensed chromatin and the particular phase could be deduced from the chromosomal arrangement. Using plasmid vectors we have shown that such assays are reliable. The cDNAs encoding these fusion proteins are being transferred to cells with lipofection, These tools could permit the detection of morphologic changes in single cells by time-lapse microscopy.

Keywords: Katushka, Sensing Cells, fluorescent proteins.