Array for the cell
After changing the research landscape for gene
expression, microarrays are turbocharging studies with
living cells
Figuring what a gene does is hard work, but it’s
vastly easier than it was a few years ago. Back then,
you would laboriously isolate a single gene, tinker
with it to get some inkling about its purpose, and then
start speculating about how it might collaborate with
other genes. Now, microarrays let researchers gather
exponentially more data about gene expression––what
each of thousands of genes does, perhaps in
coordination with other genes.
The microarray was born a decade ago as a solution
to the one-gene-at-a-time dilemma. While studying the
growth and development of Arabidopsis thaliana,
the mustard plant, Stanford University scientists created
a robotic system for screening thousands of genes at
once on small glass slides.
| Researchers in Whitehead Associate Member David
Sabatini's lab now are upping the ante with new
cell-based microarrays that promise much for the
field of drug research. |
By fixing tiny dots or “probes” (each a
short bit of DNA or RNA) onto a slide in an organized
grid, scientists fashioned a microarray, also known
as a gene chip or DNA chip. The dots were so tiny that
a slide could hold thousands of probes.
Next, a sample solution of “target” RNA
(actually a solution of DNA and RNA from broken cells)
was dropped on top of the microarray slide and held
by a slide coverslip. Each probe now served as an individual
test. When an RNA target came in contact with matching
DNA on the slide, it stuck. The target was labeled with
a fluorescent tag. The better the match, the more it
glowed. Researchers could measure each reaction and
discover which genes were active for a given target.
Just a year later, Affymetrix introduced the first
commercial microarray. In 1997, the first complete eukaryotic
genome on a microarray (baker’s yeast) was published
in Science. DNA microarrays vaulted to the forefront
of biotechnology, in turn spawning RNA-based arrays
and protein-based arrays.
Researchers in Whitehead Associate Member David
Sabatini’s lab now are upping the ante with
new cell-based microarrays that promise much for the
field of drug research. Unlike traditional cell assays,
these experiments can test thousands of living cells
at once to study cell pathways and drug interactions,
and let researchers examine how these pathways and drug
interactions affect each other.
This cell-based microarray technology “has great
potential for high-throughput screening,” says
Norbert Perrimon, a Harvard Medical School investigator
who studies gene functions on a genome-wide scale in
the Drosophila fruit fly.
A genome on four slides
The miniaturized aspect of microarrays is key to their
allure. Cell-based microarrays will let the Sabatini
lab test every gene in the entire fruit fly genome using
just four glass slides and a relatively newly discovered
phenomenon known as RNA interference (RNAi). The lab
recently proved this in principle and is now working
its way through the genome.
To use RNAi, scientists synthesize small RNA molecules
to target and “knock down” a specific gene
by blocking its ability to create protein. Each RNAi
molecule is specifically designed for its target gene,
so a 20,000-gene genome, such as the fruit fly’s,
requires 20,000 RNAi molecules.
While these molecules can be bought or generated in
the lab, the sheer volume required is a challenge. The
Sabatini lab needs significantly fewer with its microarray
format than it did with microtiter plates, a previous
technology. On a microarray, each spot needs only enough
RNAi molecules to “infect” about 300 cells.
In contrast, a microtiter plate well holds many thousands
of cells and needs a corresponding number of RNAi molecules.
Also, microtiter plates hold 384 wells each. But microarray
slides can hold thousands of samples––5,000
in this case.
Cells are then “seeded” on the array. A
solution of cells and cell growth media is poured over
the microarray slide in a Petri dish. After an hour
or two, the cells land on the bottom of the dish and
attach. A layer of cells grows over both slide and Petri
dish. Cells that land on an RNA spot probe are affected
by the probe and their reactions can be analyzed.
Plenty of groups have done RNAi arrays, notes Douglas
Wheeler, a technical assistant in the Sabatini lab,
but they’ve used mammalian cells. Those have serious
technical drawbacks, including a response to interferon
proteins (which cells produce in reaction to viral infections)
that makes cell uptake of the large RNAi molecules difficult.
Recognizing this, the Sabatini lab committed to the
same work in Drosophila. Much of the genome
is conserved between fruit flies and mammals, and the
fly cells easily take up RNAi molecules. “Because
Drosophila is so easy, why not do the initial
throwing out of the net with a screen in Drosophila,
and then do the follow-up in mammalian cells?”
asks Wheeler.
This approach should allow scientists to pin down pathways
for cellular growth, with implications for cancer research.
“We could knock down every gene in the genome
to find which genes affect how fast cells replicate
and how big they get,” says research assistant
Steve Bailey.
Faster data, faster drugs
In a second line of research, cell-based microarrays
let researchers test chemical compounds on cells by
the thousand. The more of these “small molecule”
compounds tested, the better the chance of finding one
worthy of further testing for therapeutic value. Cell-based
microarrays, which connect live cells with possible
drugs, can churn through large numbers of compounds.
In one such experiment, Bailey used a robot to print
the same 72 compounds onto a series of slides. To hold
the compounds in place, he embedded them in a biodegradable
polymer, creating a series of tiny parallel dots. “They
actually look, at the microscopic level, like Braille,”
says Bailey.
Bailey then covered the slides with cells. The polymer
slowly dissolved, and he measured how the cells reacted
to the compounds. This technique, he suggests, could
be applied to cells that mimic cancer cells. If you
combine these cells with myriad compounds, and one compound
in particular kills the cells, you might just have a
potential drug to fight cancer. The lab hopes to license
the technology.
The microarrays can do “double knockdowns,”
manipulating more than one gene at a time to see if
and how they’re connected. “One can very
quickly go through very large numbers of genes and very
large numbers of cell phenotypes and say what genes
are responsible,” says Sabatini.
“These microarrays allow use of rare cell types
(because we only need a few cells per experiment), allow
use of rare compounds, and allow imaging of cellular
functions and molecules,” adds Brent Stockwell,
an assistant professor at Columbia University and former
Whitehead Fellow who developed the microarrays jointly
with Sabatini. “They should accelerate both basic
biology and drug discovery efforts.”
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