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Sunday, October 10, 1999
To make a mouse
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TO MAKE A MOUSE
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Staff Photo by Robert Miller
Andrew Xiao gazes at a colony of bacteria that may or may not contain the gene he built with a monkey virus. If the gene is there he will transplant it into the embryo of the mouse he must make to get his Ph.D.
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Chapter 1: The Quest
In a UNC-Chapel Hill laboratory, Andrew Xiao is trying to engineer a mouse genetically susceptible to brain cancer. For cancer research, Xiao's work could be important. For the young Ph.D. candidate's future, it's crucial.
By JON FRANKLIN, Staff Writer
To get a traditional recipe for mice you have to go back to the 1600s. Jan Baptista van Helmont, the alchemist cum scientist, had a good one. You took a wad of dirty underwear and threw it in a corner. A variant of the recipe added grain. After a few days, voila! Mice appeared. Spontaneous generation, it was called.
But Andrew Xiao was not impressed. He looked up sharply from his laboratory bench, where he was attending a mixture of water, enzymes, and dissolved mouse pup toes and tail tips. He'd never heard of such a thing.
"To make a mouse," he said, "it's harder than that. To make a mouse takes a year. A year and a half. It's really very difficult to make a mouse."
He directed his attention back to the rack of tiny vials in front of him. Each contained a bit of flesh from a baby mouse. If Xiao was as lucky as he was clever, at least one of those mice would carry a brain cancer gene he had fabricated from a monkey virus.
So far, such good fortune had eluded him. He'd hoped to get his mouse in a year, but the year had come and gone and still he struggled on, his project metamorphosing first into quest and then into odyssey. Xiao's armament of knowledge and understanding seemed pathetically insufficient. Each day he faced anew the legions of chugging polymerases, slithering ribonucleic acids, and all the other stubborn little tongue-twisting robots that populated the new world that had emerged from the opium dreams of 20th century biology.
And at the heart of his enigma was the DNA molecule, master of all that lived, barricaded behind a thicket of riddles, brooding over its primordial secrets.
So if Xiao's existence was in many respects far removed from the abracadabra of van Helmont's day, his was still a sorcerer's life, full of spells and special knowledge, where recipes for mice were exchanged with the utmost seriousness and the fortunes of an apprentice like Xiao might rise or fall depending, say, on how rapidly some extract of life moved through a charged gel.
In the meantime, survival came down to rigor and focus, interminable days of study and work, endless weeks that blended together in one long agony of effort.
Not long before, Xiao had broken up with his girlfriend. She studied biology at a university several hours away and he was a Ph.D. candidate at the University of North Carolina in Chapel Hill. It was just too far. Neither had the energy to make it work.
Now it was just Xiao -- and, of course, his project.
Bending over his vials, he smiled, faintly, taking joy where he could find it. Underwear, indeed.
Xiao's mouse, if he could make it, would be different from any creature that ever lived. In the purest sense it would not be a mouse at all, or not totally a mouse because each of its cells would carry a gene alien to its murine heritage.
The gene Xiao was attempting to transplant -- the "transgene" -- came from a virus found in green monkeys. In Xiao's mouse, it should induce a susceptibility to a rare but exquisitely malignant brain tumor called glioblastoma.
The recipe had all been carefully thought through by Xiao and his mentor, Terry Van Dyke. Glioblastoma, at least in humans, usually struck in midlife. If the same was true in the mouse, then Xiao's transgenic creation would have time to procreate before it died.
Xiao would then have a line of mice that could be multiplied and shared the world over. Scientists in other institutions and other countries could study them, and perhaps make their own genetic modifications.
It didn't matter that glioblastoma was relatively rare in humans; the important thing was that it was explosively malignant. Extremely malignant cancers tended to be the easiest to understand, and the lessons could then be transferred to the more common, slow-growing forms. Research on rare lymphomas and leukemias in the 1960s and '70s, for example, underlay the '90s-era treatments for more common tumors of the breast, ovary, testicle and lung.
So while it might be unlikely that Xiao's mouse would lead directly to a cure for human cancer, the young scientist could legitimately fantasize that it would have an impact. It might prompt some other scientist, somewhere, someday, to make an observation that would trigger an insight that would lead to a procedure that would provoke a discovery that would save the lives of Xiao's own fellow human beings.
Meanwhile, and more prosaically, in the small, technical world of mousemaking, such a mouse would be quite a trick. If Xiao could pull it off it would be yet another credit to the Van Dyke laboratory.
And then there was the final matter, far from inconsequential, of Xiao's own Ph.D. and, beyond that, a good postdoc appointment ... and then a grant ... and, perhaps, some day in the dim future, a tenure-track position at a good university.
Andrew Xiao was born 27 years ago in faraway Beijing. But even as a young child he had been acutely conscious of that other, famously capitalistic nation across the Pacific Ocean. It was in the family, going back generations.
"My grandfather went to missionary school," he explained ... then hesitated, thinking back, his brows furrowing. "Or maybe it was my great-grandfather, I'm not sure. One of them. But because of that we all learned English, every generation. A lot of my family, and it's a big family, came to the West."
To preserve its Western heritage even through the cultural revolution, the family had had to create strong traditions of its own. The parents bestowed on each child a secret Western name. The firstborn male received a name beginning with "A," which is how Andrew became Andrew.
The family listened to Voice of America and the BBC, and so as a child Xiao heard, for example, the Beatles. He liked them well enough, though not so well as to buy their tapes. His ear inclined toward classical music, particularly Mozart, Beethoven and Tchaikovsky. He liked the way the masters so carefully structured the music and then layered in levels of variation to produce something that seemed deeply spontaneous. Life itself was like that.
English, meanwhile, came relatively easy to Xiao. It was the language of science and, especially, molecular biology. By the time he was in college he was reading biology in English, without bothering to translate it. Ultimately his technical English would outpace his technical Chinese to the point that he would have difficulty talking about molecular biology with a Chinese-speaking scientist.
All these things were in his favor when, in 1995, he was accepted for a master's program at the University of Maryland. But nothing could prepare him for rock music, or for Americans' constant preoccupation with material things.
At least he knew two familiar languages. English was an old friend. And the genetic code, of course, spoke in the same whispers in either hemisphere.
In America, as in China, he had concentrated on his studies and kept to his timetable. He got his master's, and came to Chapel Hill for his Ph.D. He finished his classwork, freeing up his entire life to focus on the laboratory and the next big hurdle, which was getting his mouse.
That was the thing: The mouse. He needed the mouse. It all came back, every time, to that mouse.
Xiao was aware, as he worked, of the intellectual tradition he inherited. There was a saying in science that each generation saw farther because it stood on the shoulders of its predecessors. The most fundamental law of genetics, that like begat like, had been known by ancient peoples, long before the hooded monk Gregor Mendel began tinkering with his sweet peas, long before he discovered that genetic information came in discrete packages.
Later those packages would be called "genes." But the root mechanism of genetics was not discovered until the 1950s, when James Watson and Francis Crick discovered that genes were packed inside the long, double helix molecules that Xiao would one day snip apart and patch together. The language of life had an alphabet of just four letters -- chemical bases that would become famous simply as A, C, G and T.
The scissors Xiao would use to do his tailoring were "restriction enzymes," a class of proteins designed by nature to cut other proteins in different but very specific places; the men who discovered them won Nobels in 1978. The discovery of other enzymes, designed by nature to rejoin the severed ends of DNA strands, represented the life's work of still others.
And so it was that science proceeded, generation upon generation. History dripped from every tool Xiao used, every strategy he employed, almost every thought he had. As a practical matter this imbued his work with an almost ritualistic significance, heavy with legend and scientific derring-do.
He knew his history well, but none of it weighed him down. He hadn't been born yet when Watson and Crick figured out DNA, and was but a small child when restriction enzymes were discovered. And while Xiao might be precocious, and though he was an extraordinarily focused young man, he was still a young man, and he coped with the past the way new generations always do.
From his vantage point of 27 years of life and his perch on the razor edge of molecular biology, any process older than three years was a "traditional method." If it went back more than a decade, it was "olden times."
It was a cast of mind that fit the spirit of the moment. Molecular genetics was in the throes of revolution -- like microbiology in Pasteur's day, or physics in Einstein's. Every experiment was a leap into the unknown, and every month the journals were full of yet more revelations. Anything seemed possible. At a time like this, history was not just a subject to be studied. It was something you could make.
Every day, men and women like Xiao did things that had never been done before. Sequencing new genes. Figuring out the structures of the molecules that read them. Engineering bits of DNA into virus shells, and then vectoring the result into living creatures in hopes of changing, fundamentally, what those creatures were.
It was difficult to overstate. Molecular biology had become a blur of science, engineering, art and industry; genetic information was accumulating so fast that computers couldn't keep up. The National Science Foundation had launched a special program specifically to catalog and analyze the information molecular geneticists were collecting.
The whole enterprise twisted the mind, sowed dreams of cures for disease and industries for the new century. Human enzymes and hormones, such as insulin, could be produced by other animals and made into cheap, readily available medicines. The genetics of cancer could be discovered, and with the same tools the disease could be prevented and cured. Newspapers were punctuated with stories about advances in the understanding of cancer, heart disease ... even aging.
A new world was being born, and it was happening very rapidly. Armies of molecular geneticists and biotechnologists converged on the hot spots of biology: places like Stanford University, the Massachusetts Institute of Technology, the Research Triangle. Construction crews erected steel and concrete molecular biology laboratories on university campuses; laboratory supply houses and drug companies set up shop nearby.
No one knew for sure how many people in the Triangle earned their living manipulating genetic code. But by 1999 there were a lot of them. Van Dyke, the head of the UNC laboratory where Xiao worked, guessed the number to be between 15,000 and 20,000.
In the subculture of biology, Van Dyke said, biotech workers were often known as "lab rats." She laughed. "That's probably because we spend our lives scurrying around laboratories." But she and the young scientists she trained were more likely to refer to themselves as "mousers" -- a word coined by Oliver Smithies, the grand master mousemaker, who worked nearby on the UNC campus.
Whatever their name, they did the hands-on work of the brave new world. If they lived a somewhat isolated existence, and were only dimly perceived by outsiders, the thing that set them apart was the very nature of their work: to re-engineer life.
But a young man in hot pursuit of his Ph.D. had no neurons to spare for such panoramic issues. Xiao had spent his life disciplining himself to focus on the task at hand, and never mind the revolution -- Xiao's own drama was unfolding, day by day, in the cramped and cluttered confines of the Van Dyke laboratory.
The laboratory was probably originally conceived by some architect as a single large room. But in the realpolitik of academic science, where space was money and status combined, open spaces didn't stay that way. Van Dyke's laboratory had been divided into a warren of walkways lined with black laboratory benches and, above and below, cupboards.
The space was relatively orderly, but to the uninformed eye it was a clutter of solutions, glassware and instruments. Refrigerator doors were covered with the kinds of things that occupied a genetic engineer's mind: children's drawings, jokes from American Scientist, genetic schematics of plants and animals. Scientists in white lab coats sat at their benches, focused on what they were doing, or moved, preoccupied, through the passageways. There was little noise: the distant whir of a centrifuge, the click of computer keys, soft music from a radio. And in the air, the faint but ineradicable scent of mouse urine.
Xiao's allotted space included a desk in a far corner, next to a window, and perhaps five feet of adjacent lab bench. This was where he would make his mouse and, having made it, would characterize its genetics.
It was all mapped out. It had to be. Times were tough at universities. Only one out of every four qualified postdocs was getting a faculty research job.
So he would have to be spectacular. He would have to use his mouse, or something he had learned from it, to develop a really hot line of research. That, with luck, should lead to five or six years of postdoctoral research which, in turn, should attract enough federal money to serve as a dowry when he went looking for a tenure-track position at a university.
First, though, the mouse. He had to have the mouse.
It was the way things were generally done, in his field. Certainly in Van Dyke's lab.
Yes, he said, there were cases where a young researcher like himself, through no fault of his own, had failed to produce the requisite mouse. The masters of the laboratory usually figured out some way to keep the student around a few more years, to try again and again ....
Xiao sat on his stool, elbow on the hard black surface of his laboratory bench, considering for a fleeting instant the fate of the Ph.D. student who failed to get his mouse. Then he shook his head emphatically.
"No," he said. "That would not be good. I will get the mouse."
Once, not all that long ago, it had been enough for a Ph.D. candidate just to get a transgene into a mouse and have it "take," producing a functional transgenic mouse. But as the technology advanced, the requirements ratcheted up.
Xiao's mouse was expected to be medically useful, which meant that the transgene had to be a powerful one -- powerful enough, in this case, to cause cancer.
That changed the nature of the exercise. As soon as you undertook to transplant a truly powerful gene, unexpected things started to happen. The transgenic fetus aborted before reaching term. Or it was born with grotesque malformations. Or it was quickly consumed by spreading cancer.
Such problems often had to do with differentiation, the process by which fetal cells parted company and went down separate metabolic pathways, some of them becoming, say, liver cells, and others becoming heart or brain cells. Differentiation was one of biology's central enigmas, and so the problem had the full attention of scientists.
The way life worked, each cell had a full complement of genes. Yet, once differentiation occurred, most of those genes turned off. A liver cell made only liver proteins and a brain cell made only brain proteins. And that was sometimes a problem with transgenic mice: The transplanted gene would express itself in every cell of the mouse's body. Life, at least higher forms of life, could not tolerate that.
But how did each cell know what DNA code to read, and what to ignore? The answer, when it came, followed a familiar logic -- familiar, at least, to computer programmers.
Messages sent over the Internet had "headers," which were basically address labels instructing servers where to send the code. Genes turned out to have similar headers to designate which cell type they had evolved for. The header, which appeared directly in front of the gene, said, "If you are a brain cell ..." or "If you are a kidney cell ..."
DNA was read by proteins called polymerases, which chugged up and down the DNA strand reading some genes and ignoring others. At least part of their discrimination was based on the header that preceded the gene. Different cells had slightly different kinds of polymerases, and if a kidney polymerase, for example, read a header that said "If you are a skin cell ...," then it skipped the following gene.
Biologists didn't call the "if you are" sequences "headers," however. They called them "promoters," because they promoted the manufacture of a protein. So it was that liver polymerases read genes with liver promoters in front of them, brain polymerases read genes with brain promoters in front of them, and so on throughout the thousands of different tissue types in the living animal.
By the time Xiao arrived in Chapel Hill, mousers were just perfecting their ability to manufacture specific headers and attach them to the genes they transplanted, thus ensuring that the proteins those genes coded for would be produced only in the target organ.
And that, now, was Xiao's task. The cancer gene he was using was a powerful one, and in its unrestricted form it would immediately express all over the mouse's body, and the mouse would die in the womb. That was why, to date, no mouse model of glioblastoma had been made.
He would transcend this difficulty by splicing a promoter in front of the transgene, so that it would only be read in the glial cells, in the supporting structures of the brain.
Or that, at least, was the theory that had brought Xiao to the threshold of his Ph.D.
But now, for some reason he could not seem to fathom ... the mouse eluded him.
In his childhood, Xiao and his classmates had worshipped scientists the way American adolescents worshipped rock stars. To be a scientist, if you were a Chinese boy, was to play the greatest game there was, to confront Nature herself, on her own terms, and wrest from her the secrets that would make human beings healthier and more prosperous. No calling could be higher.
In Xiao's case, it was a dream that could actually come true. His father was an electrical engineer and two of his uncles were research scientists. He could do it too. He knew he could do it, if he just worked hard enough, studied long enough, made it the priority in his life.
So he would definitely be a scientist, but he didn't know what kind until a high school teacher introduced him to the intricate, unseeable world of molecular biology. The young man was fascinated to discover that the liquids, lumps and goos that made up living tissues really weren't what they appeared to be. What the eye saw was meaningless. The eye functioned on the wrong scale.
If you wanted to understand life, you had to focus down farther than the eye could see or the retina could record, beyond the reach of the light microscope, beyond even the resolution of the electron microscope. Life proceeded on a scale so small that only one instrument could record it: the human mind.
On that scale, everything was made of molecules, which were combinations of atoms. The molecules bounced around like magnetic balls, attracting and repelling one another, and producing reactions that translated to fire and thunder, plastics and computer chips.
Most common molecules were simple. Water was composed of only three atoms, two of hydrogen and one of oxygen. But the molecules of life were huge. Many of them contained hundreds of thousands of atoms linked together in structures that folded in on themselves and became hideously complicated.
Such molecules were industrious little machines -- proteins, mostly, complete with moving parts: arms, legs, grapplers, pincers and cutters. Tractor proteins pulled loads of material from one part of the cell to the other. Others served as winches and cranes. Some welded other molecules together, fabricating new proteins, while others cut apart broken proteins to recycle their parts.
Still other molecules were, in effect, self-contained messages. Growth hormones were proteins with a particular shape; when their presence was detected by special receptor proteins on the surface of the cell, the cell began to grow and divide. Hormones of various other shapes instructed cells to stop growing, to change metabolic functions, to take on glucose fuel ... or even to die.
If a young man could focus his mind down far enough he could glimpse one of the most fundamental truths of existence ... that the very act of "living" was done by these workaholic little machines.
To learn how these machines worked was to be indoctrinated into what was for all practical purposes a secret order of scholars devoted to the contemplation of a single magical molecule: deoxyribonucleic acid. One filament of DNA strung together thousands of messages spelling out the recipe for men and for microbes, and everything in between. In recent generations these scholars, by sheer force of mind over complexity, had created a new science -- even a new kind of wizardry.
For life, being a dance of machines, was not immutable. It could be changed, redesigned, even created.
To get a grasp of what "living" meant on the scale of molecules, the apprentice wizard had to master and apply knowledge from a whole collection of other disciplines, from chemistry and physics to computer science. But it was clear early on that Xiao had what it took: intelligence, energy, vision, patience, endurance -- and, of course, desire.
The desire was an integral part of it all. For while it was true that the new biology was very complicated and mechanistic, and that it required great discipline and effort, there was also something indefinably magic about it ... and Xiao was definitely bewitched.
The decision to go into molecular biology dramatically raised the educational bar for young Xiao. As the son of intellectuals in his nation's capital city, he had been assured a good education and, eventually, entrance into a college of some sort. But if he was going to play such a high-stakes game as biochemistry, the college would have to be a very good one, indeed.
What's more, he would have to graduate at the top of his class because the next step was to get admitted to a master's program in the United States. That's where biology was happening.
In Beijing the schools were rated, as were the students, and only one out of 15 students could get into the best schools. Once in, every course was mandatory and every year was make or break, because only a fraction of each class was allowed to go forward into the next grade. After three years in high school, students took a national exam; their scores determined who would go to which college.
In this way Xiao's youth was willingly sacrificed on the altar of science. He studied very hard and, in succeeding, earned the privilege of studying harder, later. He hacked at the code of life, memorizing the names of all the little robots, the builders-up and the tearers-down of metabolism. He got the hang of thinking in nanometers and megabases and daltons.
He read and digested everything he could find about the nucleus of the cell and the library it contained -- the grammar and punctuation of DNA, and exactly how the polymerases read it.
In bacteria, the genetic material mixed freely with the protoplasm of the cell, but in more efficient creatures the DNA was walled off behind a fatty, lipid membrane -- the nucleus. Behind that membrane the double helices were tended by several classes of librarian enzymes that catalogued, read, translated and repaired the code.
When new robots had to be manufactured, the librarians read off the pertinent bit of DNA and created a ribbonlike ribonuclease, which contained the design for the robot. The ribonuclease then wormed its way through the nuclear membrane and out into the cell proper, where the robot factories were.
If this was a source of fascination to Xiao, it was also his toolbox. Scientists who re-engineered cells couldn't do so directly -- the clockwork mechanisms were too tiny, too intricate and too fast-moving. The engineers had to isolate the various robots and subvert them to their own uses.
Viruses were favorite tools because they were natural hijackers. A virus was nothing but a stretch of genetic code surrounded by a protein capsule designed to punch through the cellular membrane of its prey. Then the viral DNA commandeered the protein factories to make new virus capsules. It also insinuated itself into the cellular library, where it snipped out bits of the code that might have allowed the cell to defend itself.
For decades experts thought viruses were the smallest living things, but by the time Xiao was in high school they'd discovered a parasite even smaller. It was called a "plasmid," and it was nothing more than a loop of DNA -- pure genetic code. The only thing a plasmid could do was make another plasmid, and it could only do that inside the cytoplasm of a bacterium.
But plasmids were wonderfully malleable. DNA could easily be grafted into them and taken out again. And they were durable, so durable that they could exist outside their host cell. They were perfect for storing and duplicating genes.
Life was an amazing thing, so complex it seemed like magic, yet every process was specific, definable, learnable. It just took determination, and that was something Xiao had. He studied, he worked, he took one step at a time. And then, finally, in the fullness of time and a place halfway around the world, he was an understudy mousemaker working furiously to earn his Ph.D.
But no matter how much you knew, it was still hard to make a mouse. The recipe was complex, exacting and tedious, and had to be followed without error. The engineer could never see the object he was engineering. Everything was blind. Yet a single mistake ... or one piece of bad luck ... and it was all for nothing. So making a mouse was to make many mice, most of them rejects.
And so it went, endless, meticulous toil in the laboratory, the days coming and going in a flash, blending into weeks, the weeks becoming months, summer and fall, winter and spring. Again and again Xiao followed the recipe, varying it slightly, teasing the system, thinking that a different host mouse might help, or that slightly different procedures would produce a significantly different result. And still, yet still, no mouse.
The basic recipe used a cancer gene that came from another laboratory. To multiply it into usable quantities, he first spliced it into the DNA of a plasmid. Then he teased the plasmid into bacteria and grew the bacteria out in a flask so that the plasmids, and their transplanted gene, would multiply as well. Then he purified the plasmids and harvested the gene by adding restriction enzyme "scissors," which neatly and accurately cut the cancer genes out of the plasmid loops.
That was the first step.
The entire process took a long time. Genetic material was purified, amplified, passed through electrified gels and inserted into mouse eggs, which were in turn inserted into host mice. Days disappeared into the black hole of scientific concentration as Xiao finished one batch and started another, worried over host mice, ran genetic tests on the resulting pups. So far, all for nothing.
Oh, he had come close, all right. But never close enough.
Sometimes it seemed like bad luck followed him. He'd produced a number of transgenic mice over the past few months, but they'd all died in infancy.
The biggest heartbreak had come just a few months earlier. Xiao had isolated some viral gene fragments, and a steady-handed co-worker had injected them into the fertilized eggs collected from a line of black mice. The eggs were then implanted into the womb of a white mouse.
The good news was that the pregnancy took. The bad news was that most of the pups were born with deadly malformations of their brains. Only one tiny male survived.
Xiao had cut off the tip of its tail and one of the digits of its front paw -- enough to run a genetic test. The result was positive: The mouse carried the cancer gene.
What's more, after a few weeks' growth it became clear that it also had a brain tumor. Its movements were curtailed, and for a time it lost its hair. The gene was expressing! Extraordinary metabolic things were happening in the tiny black biosystem.
Xiao was ecstatic. He haunted the mouse facility, hovering over his creation, waiting for it to reach maturity so that he could breed it.
From a scientist's point of view the nice thing about mice was the same thing that was so awful about them from most other points of view: They bred like, well ... like mice. A female mouse could conceive at just four weeks, and 19 days later give birth to a new generation. The black mouse was a male, though, and it took males longer to mature: two interminable weeks longer.
But the time finally passed and, as soon as he could, Xiao introduced a female into the cage and waited for signs that she was pregnant.
A day passed, two. Nothing happened.
Puzzled, Xiao removed the female and replaced her with another. But more days passed, and still nothing happened.
He tried a third female. Nothing.
He checked the male over, physically. It was visibly intact, which was to say that it had testicles and there were no outward signs of malformation.
He tried another female, out of a forlorn hope, but by that time he wasn't surprised when nothing happened. He already knew, in the pit of his stomach, that the precious black mouse was sterile. It was a mouse, and he needed a line of mice. It was another failure.
What was wrong? Why did most of his mice die? Why couldn't the rest reproduce? Was he making some mistake?
There was no way to know, nothing to do except let himself be disappointed for a moment ... and then move on. To make a mouse was to make a lot of mice. He knew that.
But this many?
The next time he would just have to work harder, be smarter, make no mistakes. He would vary the recipe in some critical way. In fact, as he thought about it, he already had a glimmering of what he would do.
And then he was busy again, making another mouse.
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