Gene Guns

"What do you do when your beautiful field of barley or oats is being destroyed by an insect or a fungus? Well, in the past farmers have tried different methods~rotating their crops, trying different varieties, adding chemicals. Today here at the United States Department of Agriculture Cereal Crops Research Unit in Madison, Wisconsin they are trying a new method which makes use of a gene gun. "

Gene guns are one of the newest technologies in the exploding field of genetic engineering. Let's take a look at one specific example of gene gun use and see how it illustrates the power and the promise that will make the coming 21st century the century of biotechnology.

To understand how a gene gun works you need to know a little about genes. All living organisms, plant and animal, need a program of instructions to tell them how to grow and how to behave. That program is coded in chemical form on a long twisting molecule called DNA, deoxyribonucleic acid. Most of the DNA in living cells is in the nucleus on structures called chromosomes. Strung along the DNA of the chromosomes, like beads on a string, are sections of the DNA called genes. It is these genes that have the code that tells a cell how be a skin cell, a blood cell, a nerve cell ~ a leaf cell, a root cell, a flower cell.

When an organism, plant or animal, reproduces, the genes from the parent plant or animal are passed on to the offspring. In sexual reproduction half of the genes come from the male parent, and half from the female. The genes from each parent are not changed but they are recombined in the offspring in a new and unique way. In asexual reproduction, including cloning, the genes are also passed on unchanged from parent to offspring. Very recently biologists have found ways to not just reshuffle, but to permanently change this all important genetic information. For instance, they can change the genetic information of a fertilized egg or of a very early developing embryo by inserting genes from a different species of plant or animal. They can even put a gene from a plant into an animal, from an animal into a plant, or from a bacteria into a plant or animal, or from a plant or animal into a bacteria. When this is done, the result is what are called transgenic plants and animals. Let's see how this works with barley plants.

Barley is an important grain crop grown in northern United States and Canada as well as in northern Europe. In recent years it has been severely threatened by a mold called fusarium which attacks the seed heads, making the grain worthless for human use and consumption. At present no known variety of barley has genes that can resist this fungus. There is a gene, however, in oats that can and does resist fusarium. Why not take this anti-fungal gene from an oat plant and transfer it into a barley plant? Today the U.S. Department of Agriculture is doing just that. In a series of gene transfer experiments they hope to produce a transgenic strain of barley able to successfully resist this destructive fungus.

One of the lead scientists in this quest, Dr. Anna Maria Nuutila, explains and illustrates some of the details of how this is done.

"What we do is take a small seed here .. and dissect the embryo ... right here and we transfer the gene that we want into the little embryo with a help of a gene gun."

Gene guns are one of the new powerful tools in genetic engineering. They were first invented in 1987 for just such applications as this one. They got the name "guns" because the first models used gunpowder to propel the genes into the living plant tissues. The technology was improved in 1991 with the invention of the gene gun used today in this U.S. Department of Agriculture laboratory. It uses compressed helium instead of gunpowder as a propellant.

"This is a gene gun. This is the chamber where the actual bombardment is done and we start getting it ready for the bombardment."

Here in her laboratory she separates a number of embryos from their seed coats under a dissecting microscope. She places the embryos in a dish which will be inserted into the gene gun chamber, where they will be bombarded with a special oat gene that has information to resist fungus growth. To get it ready for use in the gene gun they must first put the oat gene into a microscopic structure called a plasmid and get the plasmid to adhere to tiny gold particles.

To do this they use a cut and paste technique.

The knife used for the cutting is a molecule called a restriction enzyme. Restriction enzymes are newly developed powerful molecules that can be bought off the shelf now from biotechnology companies. Each restriction enzyme is specialized to be able to cut a DNA strand at a particular coded spot. Once cut loose from its original DNA strand the DNA fragment a desired gene can then be "pasted" into another DNA strand with the help of a special enzyme. The DNA strand with the new gene pasted in can then be made to adhere to tiny gold dust particles which will be the "bullets" used in the gene gun.

Michael O'Connor explains how this works with the anti-fungal gene they want to put into the barley plant.

"This plasmid is actually just a circular piece of DNA where you can insert genes. .. Now how we go about this is we take the anti-fungal gene and cut it out with restriction enzymes. .. Then over here we cut a gene out of the bacterial plasmid, also with restriction enzymes.

"We can pull that gene out. This is the plasmid that we use to coat the gold dust and the gold are used in the gene gun to bombard the barley cells."

They not only put an anti-fungal gene into the plasmid but they also insert another gene to resist herbicides. This, as we will see, will give them a way to identify the plants that have the anti-fungal gene once the transfer is made. The newly cut and pasted genes in the plasmid are precipitated onto tiny gold particles attached to this thin plastic disc and now we are ready for the bombardment. Dr. Nuutila explains:

"This is a pressure disc here which we use to choose the pressure we will use in the bombardment. We are using 1100 psi. Put it into there and tighten it a little. ... This is the macro carrier on which the little gold particles have been precipitated and the little gold particles contain the DNA we want to bombard into our cells and then we turn it upside down so the little gold particles are on the bottom side now. ... The next thing we do put in this stopper screen which stops the macrocarrier particles but lets the little gold particles go through to hit the actual target.

"We close the chamber and we start taking the vacuum in. This tells us what the vacuum is in the chamber.

"Put the vacuum on hold and now we bombard as you see how the pressure goes up and then we let the air back into the chamber and take your embryos out and can put it now to grow."

The embryos grow on special culture media that has an herbicide included. Here is where the extra anti-herbicide genes come into play. The herbicide will kill normal barley cells but will not have any effect on cells that have taken up the fungal-resistant gene because these cells will also have an anti-herbicide gene. In this way they select the transgenic plants.

They grow these transgenic barley cultures on selection media for eight weeks now. By this time the little growing cells, called calli, that are transgenic are growing well, but the calli that are not transgenic are dying.

"After we have had the culture on selective media for eight weeks we can put them onto a regenerative media and on this media they start to produce little plantlets which are transgenic now."

To make sure the plantlets do have the newly transferred gene they take a sample of cells and use a newly developed technology called Polymerase Chain Reaction, PCR for short. (This is the same technology that was used to help identify blood samples in the O. J. Simpson trial a few years ago.) By carefully controlling temperature cycles the PCR machine reproduces and multiplies the microscopic DNA segments genes into quantities large enough to identify using another technology called electrophoresis.

Here we see Dr. Nuutilla adding the multiplied gene samples to the top of a special gel preparation. A small electric current is then applied. The electrical charge causes different molecules to move down the gel at different rates and with the addition of special stains we end up with a visual record of genetic composition.

Very recently they did succeed in producing their first mature transgenic barley plants. They are now producing progeny of these plants. This is easier with barley than with some crop plants because barley is self-pollinating. They hope to soon have enough transgenic seed to test in the field.

Like the PCR machine, gene guns are one of the key new instruments that are revolutionizing genetic engineering today. The gene gun is used here for barley plants. There is now available a new portable gene gun as well as the stationary one seen here. Both versions of the gene gun can be used to produce many other kinds of transgenic plants and animals today.

For instance, besides cloning the first mammal from an adult cell, the scientists at Roslin Institute in Scotland have recently produced cloned transgenic lambs. These lambs contain genes from a different species, in this case two human genes. Scientists in DeForest, Wisconsin have recently produced transgenic calves and cloned calves. How do they do this? You get cells from a sheep or cow or pig. You get genes from another species and reproduce them in a PCR technique. Now using new tools like the gene gun, or viral transfer or micro-injection, you insert these desirable genes into the sheep, cow or pig egg or embryo.

Why do they do this? You put a human insulin-making gene into a cow embryo. You engineer it so that this insulin-making gene is turned on only in the mammary glands of the cow, nowhere else. Then when the cow is milked you get not only milk but you get human insulin which you can separate from the milk and use for treating human diabetes.