Learning how to clone.

Learning how to clone.

Learning how to clone.

Health and medicine explained.
July 12 2005 9:57 AM

Caution: Student Cloner

In training on the frontier of reproductive science.

Woo Suk Hwang: a national treasure in South Korea. Click image to expand.
Woo Suk Hwang: a national treasure in South Korea

"It's not like you're flooring a car!" says Vicki Huntress, the friendly, thumb-ring-wearing lab instructor who is giving today's cloning lesson. But it's too late. The student at the microscope has pushed her foot pedal a split second longer than she should have, causing a tiny drill to penetrate too far into the mouse egg whose chromosomes she is trying to remove. The egg's cytoplasm—its vital inner contents—begins to flood out into the Petri dish, blub blub blub, a tragic scene the rest of us can see on a video screen connected to her microscope. "OK, this egg is SO going to be damaged," says the student, whose experience proved what we have already been warned: If you want to learn to clone, you are going to need a lot of patience. And a lot of eggs.

We are sitting in a laboratory in Woods Hole, Mass., where every summer the Marine Biological Laboratory, a private research institute, offers an intensive six-week course in the latest reproductive technologies. Students in MBL's Frontiers in Reproduction course are Ph.D.s and postdocs from around the world; MBL is letting me attend as well to research a book on the technology and its impact. Mornings, we hear lectures from veteran scientists in the field. We spend the afternoons practicing the lab techniques they've talked about, everything from in vitro fertilization to sperm injection to chromosome staining. Today, incredible as it still seems to me, the syllabus says: "somatic cell nuclear transfer."

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The lesson seems especially timely. Just weeks ago the U.S. House of Representatives voted in favor of federal funding for stem-cell research using excess IVF embryos, a bitterly contested measure that the Senate is expected to take up soon. The president has vowed to veto the bill if it passes Congress. Meanwhile, scientists in South Korea just achieved a breakthrough in therapeutic cloning; they created 11 stem-cell lines that are genetically matched to individual patients, an advance that highlights how far the United States could fall behind in what is shaping up to be a stem-cell version of the space race.

In theory, cloning sounds kind of easy. All you have to do is remove the nucleus from an egg (eggs, like sperm, are "germ cells," meaning they hold the reproductive capacity of a species) and replace it with the nucleus of a somatic cell, such as, say, a skin cell. (Any cell that is not a germ cell is a somatic cell.) The egg's remarkable transformative power has the ability to take that skin cell and deprogram it of its skin-cellness, erasing its hard drive and returning it to a state of "pluripotency." A pluripotent cell can be coaxed into becoming anything, including, hypothetically, a human baby. But—as has become clear during our lectures—no reputable scientist wants to clone a human baby, even assuming that it could be done, which seems dubious: Eric Overstrom, a scientist with expertise in animal cloning, talked about how hard it is successfully to clone livestock. Dolly the sheep notwithstanding, the success rates in animal cloning hover between 1 percent and 4 percent, and those few embryos that make it tend to be born with serious defects. Instead, what reputable scientists want to do is therapeutic cloning, one of the most promising areas of stem-cell research.

In fact, as we are seeing, cloning is not easy, for many reasons. One is the difficulty of obtaining the eggs, which in the case of humans must come from live female donors, something so logistically challenging and morally fraught that some scientists think it could prevent therapeutic cloning from ever becoming widespread. Another challenge—and this is our task today—is developing "micromanipulation skills." Just this morning we heard from Gerald Schatten, the University of Pittsburgh scientist who advised the Korean team, who said that one reason the Koreans are good at cloning is because they eat with steel chopsticks. He was not kidding. Cloning is like doing surgery on a dust speck. You could be the most brilliant scientist in the world, but the more relevant question may be: Did you pass "cutting with scissors" in kindergarten?

Then there are questions of technique. Currently, there is a raging international argument over the best way to enucleate. Under a microscope, eggs look like large, round cells with a gray, undifferentiated area inside the outer membrane, but within that gray area are many tiny structures, organelles and proteins and whatnot, that are crucial to embryonic development. What you want to do is remove the nuclear DNA but leave in everything else. Nobody knows the best way to do this. Cloning scientists, just now, are like chefs debating the best way to peel an onion. One popular technique is the "squish," in which the egg is turned upside down and the nucleus is pushed out. Another, "the slice," was developed by a Swiss scientist who cuts one egg in half with a razor blade, discards the half containing the nucleus, does the same thing to another egg, and squashes the two nucleus-free halves together.

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We are using "mechanical enucleation." One by one, we are sitting at a microscope to which two pipettes, or hollow glass needles, are attached. Each pipette is controlled both by a joystick, which moves the pipette back and forth and up and down, and a knob that either aspirates air into the needle or blows air out. On the floor is a pedal that causes the pipette on the right to vibrate gently and drill into the egg. Problem is, the nuclear DNA is not visible; our lab staff has helped by staining it, but the stain can be seen only under an ultraviolet light, and too much light will bleach the egg. So what you have to do is turn the light on, spot the DNA, turn the light off, and drill. Few of us are skilled enough to do all that, so Vicki is working the light for us.

Cloning is hard, but fun. One of the great things about cutting-edge reproductive technology is the way it combines sophisticated equipment with techniques that are low-tech to the point of being something a preschooler would enjoy. At the beginning of this lab unit, one of the FIR course directors, a genial scientist named David Albertini, told us that the first thing we had to learn to do was move eggs from dish to dish. Eggs, even the rodent eggs we work with, are so precious and hard to come by that one thing you do not want to do is drop the egg or lose it in your culture medium. Turns out, the most controlled way to move an egg is with your mouth. Albertini gave us each some plastic tubing, a pipette, a mouthpiece, and a filter and taught us how to make what's known in scientific circles as "egg suckers." We learned how to move eggs by sucking them out of one dish and spitting them into another. (The filter is there to prevent you from swallowing your egg.)

Today, our mouse eggs have been moved for us and placed in a dish on the stage of the microscope. When my turn comes, I manage to find an egg and secure it to the tip of my left-hand pipette by aspiration. The next challenge is to turn the egg so that the DNA is located at 3 o'clock, where the drill can get at it. "Some people like to scooch it along like this," Vicki told us, showing us how to hold the egg with one pipette and nudge it with the other. I decide to try her preferred method, which is to blow air out of the left-hand needle, let the egg float away, then catch it somewhere around 6 o'clock, a method that gradually rotates it. At first I blow too hard, and the egg zooms away. Chasing it, I learn how to blow the tiniest bit of air and catch the egg before it disappears. Next is the drilling: I drill too far, but just before the cytoplasm bubbles out Vicki turns on the light and the chromosomes are right there, so I turn the aspiration knob and voilà, the egg is enucleated!


Hey! Maybe cloning isn't so hard after all! In another room, the injection lesson is taking place. We are doing a rough approximation: In one dish are scraped-off cumulus cells, the somatic cells that surround an egg and nourish it. Our job is to aspirate one cumulus cell into the pipette, alternately suck and blow until it's literally scrambled, then inject the mushed-up cell into an egg. Sounds simple, but it's not. I do the mushing part OK but cannot get my needle lined up with my egg. The trouble is, the egg is three-dimensional—a tiny globe—but the microscope allows you to see it in only two dimensions, as though it were flat. Over and over, I try to inject, but my pipette is either above or below the egg, and the drill won't penetrate. Finally I get them lined up on the same plane, but as with cars, this foot pedal feels completely different from the other one, and before I know it I have drilled too far and out comes the cytoplasm, and with it the life of my egg.

"It's only practice," consoles an instructor, but nevertheless I feel a sense of failure. No wonder the Koreans have gone through so many eggs. In their first successful effort, announced more than a year ago, it took them almost 250 human eggs to get a single stem-cell line. In their more recent experiment, they averaged just 17 eggs per line. That's a formidable increase in efficiency. In Korea, therapeutic cloning is so enthusiastically supported that Woo Suk Hwang, the leader of the team, has been declared a national treasure. The South Korean government issued a commemorative stamp—we looked it up on the Web during a lab break—that shows a man getting out of a wheelchair and walking, then running, then embracing his wife.

Now, that may be optimistic. People still don't know if therapeutic cloning will work. People still don't know if it's safe. They don't know whether a pluripotent cell could develop into a cancerous tumor. But there's only one way to find out: practice. In this country, the climate is so hostile to practice that scientists like Schatten have to spend a lot of time trying not to run afoul of the law. Many scientists get federal funding in addition to money they might get from a private source or a state like California, and if you want to do most kinds of human embryo research, you have to be careful that no federal money goes toward that research. Schatten says the lawyers and judges he regularly consults cannot even agree on whether it is legal for him to use his lab computer to open e-mail from the Korean scientists. He is seriously considering moving some of his work to Korea. Several stem-cell researchers have left the United States for England, where limited therapeutic cloning is funded and permitted. In this country—funding aside—there is a bill in Congress that would outlaw both reproductive and therapeutic cloning. Simply put: The climate here is so confused that scientists don't know what they can do today, much less what they will be able to do tomorrow.

After Schatten's lecture, one of the students was disappointed that he spent so much time talking about politics and so little time talking about science. To me, it was hard to see how you can separate the two. That the career of every scientist in the room will be determined, in part, by what politicians decide, was one the few things about cloning that it was easy to see, even without a microscope.