A microfluidic device for temporally controlled gene expression and long-term fluorescent imaging in unperturbed dividing yeast cells.
BACKGROUND
Imaging single cells with fluorescent markers over multiple cell cycles is a powerful tool for unraveling the mechanism and dynamics of the cell cycle. Over the past ten years, microfluidic techniques in cell biology have emerged that allow for good control of growth environment. Yet the control and quantification of transient gene expression in unperturbed dividing cells has received less attention.


RESULTS
Here, we describe a microfluidic flow cell to grow Saccharomyces Cerevisiae for more than 8 generations (approximately 12 hrs) starting with single cells, with controlled flow of the growth medium. This setup provides two important features: first, cells are tightly confined and grow in a remarkably planar array. The pedigree can thus be determined and single-cell fluorescence measured with 3 minutes resolution for all cells, as a founder cell grows to a micro-colony of more than 200 cells. Second, we can trigger and calibrate rapid and transient gene expression using reversible administration of inducers that control the GAL1 or MET3 promoters. We then show that periodic 10-20 minutes gene induction pulses can drive many cell division cycles with complete coherence across the cell cluster, with either a G1/S trigger (cln1 cln2 cln3 MET3-CLN2) or a mitotic trigger (cdc20 GALL-CDC20).


CONCLUSIONS
In addition to evident cell cycle applications, this device can be used to directly measure the amount and duration of any fluorescently scorable signal-transduction or gene-induction response over a long time period. The system allows direct correlation of cell history (e.g., hysteresis or epigenetics) or cell cycle position with the measured response.
Charvin
Gilles
G
Center For Studies in Physics and Biology, The Rockefeller University, New York, New York, USA. gilles.charvin@gmail.com
Cross
Frederick R
FR
Siggia
Eric D
ED
D002453:Cell Cycle; D002455:Cell Division; D044783:F-Box Proteins; D005786:Gene Expression Regulation; D044085:Microfluidics / Q000295:instrumentation*; D008856:Microscopy, Fluorescence; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D015533:Transcriptional Activation; D043743:Ubiquitin-Protein Ligase Complexes / Q000235:genetics
D044783:F-Box Proteins; C470285:MET30 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes