Microfluidics device for single cell gene expression analysis in Saccharomyces cerevisiae.
We have measured single-cell gene expression over time using a microfluidics-based flow cell which physically traps individual yeast using microm-sized structures (yeast jails). Our goal was to determine variability of gene expression within a cell over time, as well as variability between individual cells. In our flow cell system, yeast jails are fabricated out of PDMS and gene expression is visualized using fluorescently-tagged proteins of interest. Previously, single-cell yeast work has been done using micromanipulation on agar, or FACS. In the present device agar is eliminated, resulting in a superior optical system. The flow of media through the flow cell washes daughter cells away, eliminating the need for micromanipulation. Unlike FACS, the described device can track individual yeast over a time course of many hours. The flow cells are compatible with the needs of quantitative fluorescence microscopy, and allow simultaneous measurements to be done on a large number of individual yeast. We used these flow cells to determine the expression of HSP104-GFPand RAS2-YFP, genes known to affect yeast life span. The results demonstrate inter-cell variation in expression of both genes that could not have been detected without this single-cell analysis.
Ryley
James
J
University of Texas Health Science Center at San Antonio, Sam and Ann Barshop Institute for Longevity and Ageing Studies, Department of Cellular and Structural Biology, San Antonio, TX 78245-3207, USA. james@ryley.com
Pereira-Smith
Olivia M
OM
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016922:Cellular Senescence; D003592:Cytophotometry / Q000379:methods*; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D015966:Gene Expression Regulation, Fungal; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009757:Nylons / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors; D018631:ras Proteins / Q000378:metabolism*
D001426:Bacterial Proteins; D004129:Dimethylpolysiloxanes; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D009757:Nylons; D029701:Saccharomyces cerevisiae Proteins; C085707:poly(dimethylsiloxane)-polyamide copolymer; C054562:yellow fluorescent protein, Bacteria; C070972:HsP104 protein, S cerevisiae; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins