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Harvard Team Enables Real-Time Observation of Single-Molecule Gene Expression in Living Cells


Scientists from Harvard University have developed two methods for real-time observation of single-molecule gene expression in living cells by circumventing some of the inherent problems of well-known reporter molecules, according to separate research papers published this week.

The methods are significant because they may allow researchers to obtain quantitative data on single molecular events such as protein production, as opposed to the more commonly used method of averaging together data from multiple gene expression events.

Furthermore, at least one of the methods can easily be scaled up, according to the Harvard researchers, which may eventually enable genome-wide characterization of gene expression, especially of low-copy number proteins.

The methods were developed by Sunney Xie and colleagues in the department of chemistry and chemical biology at Harvard — the culmination of an intensive four-year study, Xie said, that involved multiple postdoctoral fellows and graduate students.

The first method, which is described in the March 16 issue of Nature, is based on observing the expression of ß-galactosidase in living Escherichia coli cells with single-molecule sensitivity.

ß-gal has served as a standard reporter for gene expression for decades. However, the fluorescent products of ß-gal tend not to linger around the cell for very long, which causes a rapid loss of fluorescence despite any enzymatic amplification effects that occur.

"Many important genes — transcription factors, for example — are expressed at low levels."

Xie and colleagues got around this problem by "trapping" cells in sealed microfluidic chambers, so that the fluorescent product typically expelled from the cells could accumulate in the chambers, thus allowing the researchers to better recover the fluorescence signal.

As detailed in the paper, the microfluidic device was made of a soft polymer, and consists of a flow layer containing the cells and a top control layer. Two adjacent valves in the control layer enclose an area in the flow layer with a volume of 100 picoliters, in which the cells could be trapped and cultured.

The chip was mounted on an inverted fluorescent microscope and moved with a motorized stage, which allowed multiplexed data acquisition by repeatedly scanning the chambers. Typically, 100 chambers could be scanned in under two minutes, the researchers wrote.

"Basically you contain the fluorophores in a small volume, and the mixing is very fast, so you just monitor the accumulated fluorescence," Xie said. "As a function of time, the fluorescent signal would just go up if you have one copy of the enzyme, because you would just keep cranking out fluorescent molecules."

The real trick, however, to measuring single-molecule protein expression, Xie said, was to observe abrupt jumps in the fluorescence output, which corresponded to "bursts" of protein production. It turns out that the size of these bursts corresponded to the number of protein molecules being produced at a given time.

The distribution of protein molecules generated per mRNA has been theorized, Xie said, but never experimentally determined.

The second method for single-molecule measurement of gene expression is detailed in the March 17 issue of Science. In this method, Xie and colleagues monitored real-time expression of fluorescent reporter protein constructs in E. coli cells.

"It's akin to the ability to listen to conversations in a room full of people all talking at the same time."

Specifically, they used a variant of yellow fluorescent protein, called Venus, as the reporter, because it has a short maturation time. However, it is difficult to image a single fluorescent reporter molecule in a cell due to fast diffusion of the fluorescent signal through the cytoplasm.

To avoid this problem, the researchers designed a fusion protein consisting of Venus and a cell membrane protein called Tsr, and used the construct to monitor the activity of the lac promoter.

In this case, the researchers used an epifluorescent microscope coupled with a CCD camera to image the Venus construct. To count the fusion proteins as they were generated, Xie and colleagues photobleached the fluorophores following their detection. They found that in each image, one cell typically produced no more than five fluorescent protein molecules, which could then be spatially resolved from one another.

Both methods may allow researchers to explore gene expression in a way that has not been feasible previously.

According to Xie, the dynamic range of gene expression goes from 10-2 copies per cell — or less than one, meaning on average there may not even be a single copy of a protein in a cell — to 108, where a massive number of protein copies exist.

"If the number is 108, you don't need to do any of this — you run gels, or columns," Xie said. "If it's somewhere around 103, you can do mass spectrometry, or you can do microarrays. Our technique is able to fill the gap between the low copy number and intermediate copy number proteins. Many important genes — transcription factors, for example — are expressed at low levels."

In addition, Xie said, low copy number isn't the only factor — there is a need to be able to determine the distribution of proteins within a single cell.

"In conventional techniques, if you have many, many cells, you can make sensitive enough measurements, but you only get the mean," Xie said. "However, we can get the distribution with single-cell analysis.

"I should stress that people have been doing single live-cell measurements for quite some time," he added. "What's unique here is that they are single-molecule measurements within single cells."

According to Xie, the ß-gal method is the most conducive to scaling up to the level of high-throughput whole-genome expression studies of low copy number genes, but the YFP method may also be useful on a larger scale. The Harvard researchers were also able to apply the ß-gal technique to budding yeast cells and mouse embryonic stem cells, demonstrating its generality.

"There has been a lot of effort put into trying to develop single-molecule methods for this," Jeremy Berg, director of the National Institute of General Medical Sciences and co-chair of the NIH Director's Pioneer Award, told CBA News. "From my perspective, it's akin to the ability to listen to conversations in a room full of people all talking at the same time.

"If you're looking at gene expression, for example, a typical measurement is made on thousands of cells or more, and you measure a total increase in RNA level," he added. "But if you're really interested in fundamental mechanisms of transcription control — or in the long run, more complicated regulatory pathways — being able to do this at the single-cell level is really going to allow people to test ideas about how things really work."

The Harvard researchers are not currently thinking about commercializing either technique, Xie said.

"It's not impossible, but it needs future work," Xie said. In particular, although the microfluidics component of the ß-gal method has been worked out fairly well, the molecular biology still needs to be perfected and further tested.

Much of the funding for the project outlined in the Science paper was made available because Xie was named one of nine inaugural NIH Director's Pioneer Award recipients in September 2004, which is part of the NIH's Roadmap for Medical Research initiative. The work in the Nature paper was funded by the US Department of Energy's Genomes: GtL program.

The NIH Director's Pioneer Awards are an attempt to "support individual research that is higher risk and higher impact," NIGMS' Berg, said. "This is the kind of research result everyone involved in the program had in mind," he added.

— Ben Butkus ([email protected])

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