News of the WeekNUCLEAR TRANSFER

Misguided Chromosomes Foil Primate Cloning

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Science  11 Apr 2003:
Vol. 300, Issue 5617, pp. 225-227
DOI: 10.1126/science.300.5617.225

While governments around the world debate how to prevent human reproductive cloning, it seems that nature has put a few hurdles of its own in the way. On page 297, a team reports that in rhesus monkeys, cloning robs an embryo of key proteins that allow a cell to divvy up chromosomes and divide properly. Unpublished data from this and other groups suggest that the same problem may also thwart attempts to clone humans.

There are potential ways around the newfound obstacle, but for now, groups that made controversial claims that they would use the techniques that produced Dolly the sheep to create human babies are unlikely to succeed.

It is almost as if someone “drew a sharp line between old-world primates—including people—and other animals, saying, ‘I'll let you clone cattle, mice, sheep, even rabbits and cats, but monkeys and humans require something more,’” says Gerald Schatten of the University of Pittsburgh School of Medicine, a leader of the rhesus monkey study.

Schatten and his colleagues have tried hundreds of times to clone monkeys, only to fail. Indeed, although several groups have attempted it, no one has yet produced a monkey through somatic cell nuclear transfer, the process by which a nucleus from one cell is extracted and injected into an egg whose own nucleus has been removed. “The failure to clone any primate has so far been startling,” says Rudolf Jaenisch of the Massachusetts Institute of Technology in Cambridge, who studies cloning in mice.

The scientists had suspected for several years that something was disturbing cell division in cloned embryos. The embryos seemed normal at their earliest stages, but none developed into a pregnancy when implanted. When the researchers looked more closely, they realized why: Many of the cells in a given embryo had the wrong number of chromosomes. Some had just a few, whereas others had twice as many as they should. Although embryos can survive for a few cell divisions with such defects, soon the developmental program becomes hopelessly derailed.

To find out what was interfering with proper cell division, the team fluorescently labeled the cell-division machinery. The cells' mitotic spindles, which guide chromosomes to the right place during cell division, were completely disorganized. And two proteins that help organize the spindles, called NuMA and HSET, were missing.

A look at unfertilized rhesus oocytes explained why. The team found that the spindle proteins are concentrated near the chromosomes of unfertilized egg cells—the same chromosomes that are removed during the first step of nuclear transfer. In most other mammals, Schatten says, the proteins are scattered throughout the egg, and removing the egg's chromosomes seems to leave enough of the key proteins behind for cell division to proceed.

Missing in action.

In human embryos, as in this egg fertilized by two sperm (red lines), proteins from egg and sperm combine (yellow) to guide cell division. Embryos formed by nuclear transfer lack these proteins.

CREDIT: G. SCHATTEN/UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE

The work “explains why no one has yet succeed in achieving normally developing embryos from human nuclear transfer,” says Roger Pedersen of the University of Cambridge, U.K., who attempted human nuclear transfer experiments at his previous laboratory at the University of California, San Francisco. “Primate eggs are biologically different.” Schatten says preliminary data suggest the proteins are also concentrated near the nuclear material in unfertilized human eggs.

A cloning lab might surmount the hurdle, says Schatten, by reversing the order of the traditional nuclear transfer procedure: First add an extra nucleus, then activate cell division, and finally remove the egg's DNA. The find “will make people think differently about the optimum sequence of nuclear transfer procedures,” says Ian Wilmut of the Roslin Institute in Midlothian, Scotland, a leader of the team that cloned Dolly.

Even if scientists could overcome the obstacles, however, another study suggests that further developmental problems threaten clones of all species. Jaenisch and his colleagues report in the 15 April issue of Development that genes important to early development frequently fail to turn on in mouse embryos cloned from adult cells. That failure helps explain the low survival rate of such embryos, Jaenisch says. But he notes that the team's work—which examined the expression of just 11 genes—is only the tip of the iceberg. In other experiments, the researchers have found that even apparently healthy cloned mice show abnormal levels of gene expression. “There may be no normal clones,” Jaenisch says.

Although revising the nuclear transfer procedure might help solve the cell-division problem, it is harder to imagine a solution for the faulty gene regulation that Jaenisch and his colleagues see. “We're looking at a more fundamental problem,” he says.

The biological roadblocks would seem to be good news for those worried about the ethical implications of human cloning, says Schatten. “This reinforces the fact that the charlatans who claim to have cloned humans have never understood enough cell or developmental biology” to succeed, he says. The debate will go on, but nature already seems to have imposed its own limits on cloning.

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