Microsurgery on fruit-fly embryos

The fruit fly is one of the most important organisms used in genetic research, which can offer us insights that will help to cure disease.

Fruit flies are ideal for genetic studies, but handling microscopic fly embryos demands technology at micro and nano level.

By Ralph W. Bernstein & Åse Dragland

Fruit flies are insignificant-looking 3 mm-long insects that thrive best on rotting fruit. Since scientists began to study them nearly 100 years ago, they have become one of the most important species used in biological research. The flies are small and easy to handle, have short development and life-cycles, and are cheap to maintain in large numbers. Furthermore, a large number of genetically modified strains of fruit fly are available for scientific purposes. The fruit fly has one of the most intensively studied genomes in the animal kingdom, which was "completely" mapped in 2000. A fruit fly possesses some 14,000 genes, while a human being has 70,000.
Genetic modification of fruit flies has given us insights that are not scientifically interesting but can also help to cure disease. The fact that this type of research also has human health-care applications was confirmed by the award of the 1995 Nobel Prize in Physiology or Medicine to Ed Lewis, Christiane Nusslein-Volhard, and Eric Wieschaus for their ground-breaking work on fruit flies.

Microinstruments
This forms the background for a three-year research project at Stanford University, which is being funded by the Defense Advanced Research Projects Agency (DARPA), and which aims to improve the methods used in this field of research. The idea is to utilize microtechnology to improve experimental efficiency and control, and to develop microinstruments for handling, injecting, and finally sorting the tiny fruit fly embryos.
One of the authors (Ralph W. Bernstein) was spending a year's sabbatical at Stanford, and was currently a member of the project research team, which is part of the Photonics Laboratory led by the Norwegian-born Professor Olav Solgaard.

OMicrocannulae (left) and traditional glass cannulae used to penetrate embryos. The microcannulae are produced by surface micromachining of silicon nitride. The cannulae are less than five microns high and may be almost as narrow as five microns. The needle shown here is 30 microns in diameter and causes little injury to the embryo. The broken surface of the tip of the traditional glass cannula (right), on the other hand, is extremely irregular and not particularly sharp. The diameter of such glass cannulae is very variable, since they are produced by drawing them out by hand.

Microinjection
One important method utilized in developmental biology is the injection of substances that affect the make-up of a cell or an organism. Microinjecting such transgenes, i.e. DNA structures that often consist of a gene and a control component, results in fruit flies with new characteristics, since the transgene is integrated into the fly's own DNA. This makes it possible to determine which genes are important for the development of the organism and which organs are affected.
A fruit fly embryo is less than half a millimetre long and a quarter of a millimetre in diameter. Embryos are usually injected using microcannulae which are inserted at the single-cell stage or into a fluid-filled vacuole when the embryo is still at a very early stage of development (the blastocyte stage).
At present, such cannulae are made by heating a glass tube which is then drawn out by hand until it is sufficiently fine. The embryos are then injected one at a time under the microscope, an extremely time-consuming task. One disadvantage of glass cannulae is that their dimensions can be highly variable, leaving the scientist with little ability to control the quantity of material he is injecting. There are also limits to just how narrow such cannulae can be made. This has implications for the sort of experiments that can be done and for the amount of injury that they cause the embryo.

High-tech microcannulae
The first thing that the project group focused on was the development of cannulae with the aid of microtechnology. The cannulae are now produced by surface micromachining of silicon. Production is based on the same fabrication processes as are employed to manufacture microprocessor chips and other types of microelectronic components. As well as the processes used in the manufacture of standard microelectronics, we have developed micromachining techniques that allow channels, cavities, membranes, beams, and other three-dimensional structures to be made. The cannulae that the research team is now capable of producing are less than five microns deep and can be almost as narrow as five microns. This enables the cannula to be inserted into the embryo without causing too much injury. The use of microtechniques of this sort gives us much better control of the dimensions of the cannulae and thus of the quantity of material being injected.

More than just miniaturisation
This type of micro- and nanotechnology opens up a number of new possibilities. As well as miniaturization itself, it is suitable for the high-volume, low-cost production of microcomponents. Perhaps even more important is its potential for producing two-dimensional matrices that contain a large number of completely identical microelements. Scientists working at Stanford wish to exploit this aspect in order to make a large number of injections in parallel by using as many as several hundred microcannulae. The figure shows a sketch of such a system.
Experiments of this sort will also be dependent on being able to handle large numbers of embryos with a high degree of precision. Among the strategies adopted to achieve this is to develop a microsystem in which embryos will be handled individually in microchannels by means of a flow of liquid controlled by microvalves and micropumps. But the research group is also working on technology for fixing the embryos in particular positions, so that the injectors will all act precisely where the scientists want them to do so. This will be done by special surface treatment of a gold patterned silicon or glass wafer. When the embryos are flushed over this structure, they will be immobilized specifically at the gold sites.

Ralph Bernstein of SINTEF Electronics and Cybernetics has been on a year's sabbatical at Stanford University. He is a member of the microtechnology research team.

Many possibilities
The Stanford University team is about halfway through the three-year project, and we have demonstrated that such microchannels can be used to inject fruit fly embryos, but no active biological material has been injected so far. We have also developed a series of techniques for determining and monitoring the quantity of material injected. A microchip for sorting embryos has also been demonstrated, and we have managed to immobilize embryos.
The next stage will be to improve existing experiments, and the research team has made a good deal of progress in this respect. The long-term goal is to make it possible to perform experiments that cannot be done using traditional techniques, e.g. to make injections into individual cells after the embryo has begun to develop. This will mean producing microinjectors with diameters of less than five microns.

What is being done in Norway?

Some groups in Norway are also working on embryological research of this sort. Professor Andrew Lambertsson of the Dept. of Molecular Biology in the University of Oslo, for example, is working on injecting fruit fly embryos. Sigurd H. Fromm, a senior lecturer in the Dept. of Oral Biology's Molecular Embryology Laboratory, has been working on the topic of injection for several years, particularly on mouse embryos. He mentions that his research is not simply a matter of understanding the development of an organism. For the past 15-20 years, microinjection into zygotes of "designer" DNA structures or transgenes has played a decisive role in the development of modern biomedicine, for example in our understanding of the development and treatment of various types of cancer. Fromm's group began producing transgenic mice in 1984, and to date has established well over 1,000 "founder mice", having collaborated for a long time with Astra AB in Sweden.

As is well known, a national microtechnology research programme is under way in Norway, including the establishment of the Norwegian Microtechnology Centre in Gaustadbekkdalen in Oslo. As part of this national effort, the Research Council of Norway, SensoNor ASA, Norchip, and SINTEF have financed Bernstein's stay at Stanford. As well as doing research in the fields of microtechnology and biomedicine. Bernstein is also forging links with, and studying the efforts of, research groups in the USA.