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  • Fluorescence flow cytometry monitors gene expression in time and space

Aug 2007
David L. Shenkenberg

If you look at an unfinished painting, you might wonder what the artist was trying to express before he set aside his paintbrush. Techniques such as fluorescence microscopy and microarray analysis can provide information about gene expression, but neither provides a complete picture. High-throughput fluorescence profiling using flow cytometry is a complementary approach that enables analysis of entire gene expression patterns within small organisms. Therefore, researchers at Dana-Farber Cancer Institute in Boston have used the method to observe gene expression in time and in space in GFP-expressing Caenorhabditis elegans.

The researchers used a flow cytometer from Union Biometrica Inc. of Holliston, Mass., which can accommodate objects the size of the C. elegans worms. It automatically analyzed about 100 worms per second, a large advantage over slower and manual methods such as microscopy, said postdoctoral fellow Denis Dupuy of Dana-Farber. The device uses a puff of air to separate the organisms, a safer method than electrostatic sorting.

They were joined on the study by scientists from the University of Connecticut Health Center in Farmington, the University of Leeds in the UK, the University of British Columbia in Vancouver, and Simon Fraser University in Burnaby, British Columbia, who provided various GFP-expressing C. elegans.

They used approximately 900 genetically distinct worms. In each worm, GFP fluorescence reported the expression of a single gene. In other words, the scientists studied about 900 worm genes, representing about 5 percent of the entire genome. “The idea is to get a global view of gene expression by looking at a large number of genes,” Dupuy said.


Researchers analyzed both the time and location of gene expression in C. elegans worms, which corresponded to GFP expression. Images reprinted with permission of Nature Biotechnology.

He also said that they studied C. elegans because the worms and humans have genes with similar sequences, and those genes could have the same function. Therefore, the timing and location of expression of these genes in C. elegans could occur in a similar pattern in humans. In particular, C. elegans often is employed as a model of organ development.

Using flow cytometry, the researchers observed GFP expression across the length and width of the worms over time, and they discovered that the genes expressed together in time and space have similar functions. They were surprised to find that the expression is highly tissue-specific, Dupuy said. Finally, by comparing the flow cytometry data with known interactions among the proteins in other organisms, they learned how the proteins probably interact.

Close-ups of each segment of the worm show precisely where and when specific genes are expressed.

Dupuy said that the latest version of the flow cytometer allows for simultaneous use of three different markers. Thus, they could use a marker to determine the orientation of each worm’s head and tail, which can be difficult to distinguish, or they could directly label tissues involved in gene expression.

Because flow cytometers also are available for Xenopus, Drosophila and zebra fish, Dupuy noted that it theoretically would be possible to conduct similar experiments in other organisms, but he said that it would be more difficult.

Nature Biotechnology, June 2007, pp. 663-668.

fluorescence microscopy
Observation of samples using excitation produced fluorescence. A sample is placed within the excitation laser and the plane of observation is scanned. Emitted photons from the sample are filtered by a long pass dichroic optic and are detected and recorded for digital image reproduction. 
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