Press
Release 20 March 2003 [PDF]
[Deutsch]
One
of the most powerful tools in today's biological and medical
science is the ability to artificially remove and add bits
of DNA to an organism's genome. This has helped scientists
to understand problems caused by defective genes, for example,
which have now been linked to thousands of human diseases.
So far the technology has been limited to small segments of
DNA. But four years ago, Francis Stewart and his colleagues
at the European Molecular Biology Laboratory [Heidelberg]
developed a new technique to engineer greater stretches of
DNA in bacteria. The researchers, now working at Biotec –
Technical University in Dresden, have just used this method
to engineer a complex set of changes in a mouse gene, in hopes
of shedding light on human leukemias. Their work appears in
the current edition of the journal Nature Biotechnology.
Over two decades ago, researchers learned to use bacteria
as "copy machines" for DNA taken from other organisms. This
was a huge step for biotechnology, because most types of research
require billions of copies of a molecule under investigation.
However, there was a limitation: researchers need to change
the DNA molecules in precise ways and for large molecules
– such as whole genes – this was tremendously difficult.Stewart and his colleagues thought that bacteria could be
taught to do better, so they "borrowed" a strategy that organisms
such as mice and yeast use to repair breaks in DNA. Proteins
called recombinases circulate through their cells, looking
for loose DNA fragments that have familiar sequences. "Recombinases assume that the fragments have been cut out
of the DNA by mistake, so they try to glue them back into
the genome in the right place," says Giuseppe Testa, who headed
the current study. "Sometimes they're a bit over-industrious;
they put in pieces that look right, such as variations of
a gene that have been put into the cell by a researcher." Called homologous recombination, this process works a bit
like a "find-and-replace" command in your word processor.
Imagine you have typed "Stephen Q. Gould" everywhere, and
suddenly discover that the middle initial should be "J". The
computer can be told to look for "Stephen" and "Gould" and
replace what comes between them. In the same way, recombinases
find recognizable sequences of DNA to the left and right of
a target and replace what comes in between with the new sequence. Homologous recombination was known to occur in bacteria, but
it hadn't been possible to use it to engineer DNA, as was
the case in yeast and mouse stem cells. Stewart's team decided
to try to find a strain that could do it. "We ordered as many
types of E. coli as we could, looking for defects in the way
they repair their DNA," he says. "After five months of work,
Youming Zhang, a postdoc in the lab, found the strain." The group quickly identified the bacterial factors involved
and turned them into a new tool called Red–ET recombination
that is now being adopted by biologists all over the world.
Red–ET recombination is proprietary technology of the EMBL
and is one of the mainstays of Gene Bridges GmbH, an EMBL
spin out company that Stewart and his colleagues founded in
2000 to develop the commercial implications of the breakthrough.
The technology is licensed exclusively to Gene Bridges by
EMBL Enterprise Management GmbH [EMBLEM], the subsidiary and
commercial arm of the EMBL. "We have been pushing it to work with larger and larger bits
of DNA," Testa says, "and our latest project has been to engineer
an entire artificial chromosome in bacteria. We've constructed
a large, complex 'cassette' that we've now inserted into a
mouse in place of its normal gene." The gene that they chose is called mixed-lineage leukemia
[Mll], and is known to become defective in childhood leukemias
in humans. By inserting the artificial version into the mouse,
researchers hope to understand how the defects lead to disease. "There are many things that can go wrong in this gene," Testa
says, "and we wanted to construct a version of it that would
allow us to test as many aspects of the problem as possible." The artificial Mll that they have put into the mouse will
permit a variety of experiments. It contains two defects in
the genetic sequence that have been linked to leukemias. The
cassette also contains control switches that allow each defect
to be "switched on" whenever the researchers choose; they
can also be left off. "We can study each mutation independently,
or watch how they act together, or control the time at which
each one acts," Testa says. "This will give us a new look
at subtle relationships between multiple defects." Many diseases are linked to single mutations; however, disease
susceptibility also often relies on other sequence variations,
known as polymorphisms, in the human population. "The Mll
cassette shows, in principle, a simple way to study both a
mutation and a related polymorphism in the gene of interest,"
says Testa. "This aspect of making mouse modelswill become
increasingly more important for authentic modeling of human
disease susceptibility and the way organisms respond to drugsand
we think that our work shows the way to set up these models."
The new work also heralds a new era for genomic
engineering in many living systems. "The Mll
cassette is a first demonstration of what can
be done with large DNA molecules," Stewart says.
"Red–ET recombination increases the size
of DNA that can be comfortably engineered by
more than ten times and opens up new possibilities
for genomic engineering that will filter into
standard practice in the next few years." Source Article Engineering the mouse genome with bacterial artificial chromosomes to create
multipurpose alleles
Giuseppe Testa, Youming Zhang, Kristina Vintersten, Vladimir
Benes, W.W.M. Pim Pijnappel, Ian Chambers, Andrew J.H. Smith,
Austin G. Smith and A. Francis Stewart
Published online Nature Biotechnology 10
March 2003, doi:10.1038/nbt804
Scientific
Contacts Francis
A. Stewart
Technical University Dresden, c/o Max Planck Institute for Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307 Dresden, Germany
Tel: +47 [0] 351 210 2691
Fax: +47 [0] 351 210 1409
E-mail: stewart@mpi-cbg.de
Giuseppe Testa
Technical University Dresden, c/o Max Planck
Institute for Cell Biology and Genetics, Pfotenhauerstrasse
108, D-01307 Dresden, Germany
Tel: +47 [0] 351 210
2723
Fax: +47 [0] 351 210
2020
E-mail: testa@mpi-cbg.de |
