By David N. Leff
Now that TV quiz shows - such as "Greed" and "Who wants to be a millionaire?" - have surfaced on prime time, try this multiple-choice challenge:
When was the first animal cloned?
(a) in the 1990s?
(b) in the 1980s?
(c) earlier in the 20th century?
(d) in the 19th century?
(e) in the 18th century?
The correct answer, according to cell biologist Gerald Schatten, is "(e)." Unlike the nuclear transfer approaches practiced by cloners in the 1990s and 1980s, the earlier strategies employed embryo-splitting techniques.
"The embryo-splitting idea is not new," Schatten observed. "It was done two centuries ago with sea urchins and frogs. In the late 20th century it was done with mice, sheep, cows - even with human embryos in 1993. But not with subhuman primates - until 1999."
The current issue of Science, dated Jan. 14, 2000, reports: "Clonal propagation of primate offspring by embryo splitting." Schatten, the paper's senior author, heads the laboratory of Assisted Reproductive Technology at the Oregon Regional Primate Research Center in Beaverton.
"Our contribution here," he told BioWorld Today, "is showing that primate embryos can be split, and will go to term - after 5.5-month gestation." He and his co-authors have achieved the birth last September of the first nonhuman primate clone produced by embryo splitting. They named this healthy female Rhesus monkey "Tetra."
Schatten compared Tetra with Dolly, the cloned ewe. "Dolly and her sisters," he pointed out, "are all identical in terms of nuclei. But they all come from different egg cells, so in terms of mitochondria and cytoplasm, they're chimeric. In contrast, when you split an embryo, you have both nuclear and cytoplasmic identity.
"I think there are three major biotechnological issues here," Schatten said. "One, in a sense, bridges the gap between mice and people. The second highlights the embryonic stem cell potential, and the third separates the genes from the environment for so many of the cloning experiments."
Enlarging on the mouse-human dichotomy, he pointed out that, "With the Human Genome Project coming into reality, there may be a requirement for an animal model that is closer to humans than transgenic mice."
The second of his three potential payoffs, Schatten went on, "is the exciting promise of stem cell research. But the unknown factor here is whether it's fair to extrapolate from inbred mice - in which stem cells work, but only with certain strains - to the fully outbred population of humans. In fact, one might even question whether the basic work should even be done on human embryonic stem cells, which are coming from infertile couples at in vitro fertilization clinics, but who are not there because they are interested in embryonic stem cells."
Prime Primate Stem Cell Source
Schatten's solution to this drawback, he said, "is the ability to make stem cell lines in a fractioned embryo, tested in a monkey that is genetically identical to the stem cells. Because if those lines show full totipotency, there's every reason to say, 'Wow! This is a terrific green light for stem cell therapies.'"
The third issue on Schatten's agenda, he said, "means that using genetically identical monkeys would lead to some of the cures that we need today." He counted some of the ways: "For example, with identical twins you could evaluate the efficiency of a gene therapy vector, the safety of the newest schizophrenia or obesity drugs, and whether some of these HIV vaccines are really going to show promise.
"Also," he continued, "you could evaluate the role of the maternal environment." He cited a recent finding that a low thyroid hormone level in mothers during pregnancy can influence the I.Q. of their babies by as much as a 10-point drop. "But it's not a genetic drop," he explained. "It's epigenetic - caused by some environmental influence, such as maternal alcohol or nicotine intake.
"For example," Schatten suggested, "you could have three embryos, each carried by a different mom. Maybe one listens to Mozart, one to heavy metal and one to stock market reports." (In fact, there's a conceptual similarity between stock splitting and embryo splitting.)
He recounted how embryologist Anthony Chan, the Science paper's lead author, performed the embryo splitting, leading to Tetra's birth - and name: "First, Chan took eggs from donor monkeys and fertilized them in vitro. Then, he removed the zona pellucida - the jelly-like coat surrounding 107 embryos at the eight-cell stage, and dissolved the glue between the cells.
"Next, Chan gently teased the eight cells apart into individual cells, aspirated two of the cells into a very fine pipette, then injected them into a donated, empty jelly-coat egg shell that we got from other monkeys - and some from cows, which also worked. He repeated that three more times, ending up with a set of four tetrad embryos, each composed of two cells from the one original eight-cell embryo.
"Finally, Chan transferred two of those embryos to one foster mother and the other two to another female. Each became pregnant. One had a miscarriage, and the other, after a normal gestation of 157 days, gave birth to Tetra.
Success But No Cigar - Yet
"Since then," Schatten continued, "we've gone back to identical twins, not quadruplets. And of the last seven transfers we've done, we have four pregnancies - a success rate of about 57 percent. The last four that I mentioned are gestating as we speak. They all seem perfectly fine. Romulus and Rhesus will be born in mid-May."
He and his co-authors "are excited about that 57-percent success rate," Schatten pointed out, "because if in vitro fertilization normally results in a greater than 50-percent pregnancy level, nonhuman primate reproduction by split-embryo cloning becomes interesting, but only if it also results in a similar success rate."