Fluorescence Studies of Carbon Nanotube Uptake by Saccharomyces cerevisiae

Posted July 25th, 2011 at 7:00 pm.

Abstract: Alexandra Raeber

Mentor: Silvia Porello

Methods for introducing DNA or other molecules into cells have recently been the subject of a great deal of research because they are a necessary aspect of gene therapy and useful for drug delivery. One of the methods currently being studied is the use of carbon nanotubes (CNTs). While the insertion of CNTs into plant and mammalian cells has been studied, their interaction with yeast cells has not. Because of its biochemical and structural similarities to other, more complex eukaryotic cells, including human cells, yeast provides an interesting system for the study of CNT uptake and toxicity. In addition, yeast has many technical advantages, including rapid growth, dispersed cells, low cost and well-defined genetics. Yeast is also a possible target for CNT applications in the treatment of fungal infections.  Prior research by this lab group has shown that single walled CNTs (SWCNTs) that are functionalized with a green fluorophore (FITC) are capable of insertion into yeast cells.

The primary goal of this of summer’s research is to study the method of uptake of CNTs by yeast cells using fluorescence microscopy and flow cytometry. Fluorescence microscopy can show whether or not the tagged nanotubes are localized within the cells or outside them. Flow cytometry will be used to compare the total n umber of cells in a sample to the number of fluorescent cells to determine the percentage of cells that have incorporated the CNTs. Treating the cells with an endocytosis inhibitor will prevent uptake of CNTs and thereby abolish fluorescence within cells if CNTs are being incorporated via endocytosis. If CNT uptake is occurring through a different mechanism, fluorescence will be observed.  Conducting toxicity studies is a secondary goal.  Recent research has raised concern over the possibility that CNTs may cause DNA damage within cells1. DNA damage can be studied by looking at the concentration of DNA repair enzymes that are present in the cell, including OGG1 in yeast.  Western blotting will be used to determine the level of OGG1 or another DNA repair enzyme in yeast cells that have been incubated with CNTs versus those that have not.

References

  • Zhu, L.; Chang, D. W.; Dai, L.; Hong, Y.DNA Damage Induced by Multiwalled Carbon Nanotubes in Mouse Embryonic Stem Cells Nano Lett. 2007, 7, 3592– 3597
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