By David N. Leff

Bossy, the gentle dairy cow, and her snorting bullish consort, are bovines that answer to the name Bos taurus. Their earliest living ancestor is Bos gaurus, the gaur (rhymes with flower), a wild ox that inhabits the hilly forests of India, Indochina and the Malay peninsula.

Despite its 1-ton size - 6 feet or higher at the shoulder - and widespread, wickedly curving horns, the gaur is no match for Homo sapiens. People shoot B. gaurus for its meat, horns, hooves, hides - and for sport. More insidiously, expanding human populations keep encroaching on its wilderness habitat.

An estimated 36,000 gaurs still hang on in the Southeast Asian wilds, and some 200 live out their lives in zoos.

In short, the gaur is an endangered species - so listed by the World Conservation Union. One scientist among the many who don't want to see gaur vanish from the earth is reproductive physiologist Philip Damiani, a research physiologist at Advanced Cell Technology Inc. (ACT) in Worcester, Mass. He is senior author of a paper in the October 2000 issue of the journal Cloning. Its title: "Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer."

"This is the first study," Damiani told BioWorld Today, "to have proven that we can use eggs from one species to clone another species, and get normal fetal development." This was the same approach that ACT used last year to deliver six healthy Holstein calves, cloned from senescent somatic cells. (See BioWorld Today, April 28, 2000, p. 1.)

Damiani recited, step by step, how he and his co-authors applied this technology to jumping the species barrier between domestic cows and wild gaurs:

"The technique involved taking a germ-line egg from a domestic cow, removing the DNA from it, then taking a somatic cell from the endangered gaur, and electrofusing its nuclear DNA into the cytoplasm of the cow's egg. That allowed us to use a large number of domestic cow eggs as recipients for the endangered species' cell nucleus, or DNA."

From 692 Starter Egg Masses To One Final Fetus

"Once those fused cells produced embryos," he continued, "instead of transferring these back into endangered gaur females - of which there were few to work with - we thought that, because the 692 pre-embryonal dividing egg clusters originated from a cow egg, transferring them back into a cow would be a more logical step. So we transferred 42 of the 81 maturing embryos - blastocysts - that were produced into 32 domestic cows in estrus, as surrogate mothers.

"When we started detecting pregnancies," Damiani went on, "we sacrificed three fetuses [two of them twins] to evaluate for normal development, and also prove that these fetuses we collected were actually gaur-derived DNA, as opposed to cow DNA. That assay showed that indeed, all the fetuses we harvested indicated that the gaur DNA was able to reprogram itself in cow egg cytoplasm, and allow for normal development to occur."

However, those multiple nuclear transfers were fraught with further attrition. Only eight of the 32 blastocyst-impregnated cows went on to become pregnant. Then in the first trimester of normal nine-month gaur gestation, four growing fetuses spontaneously aborted. And in the final trimester, an unexpected, late-term miscarriage occurred. The body weight of this nearly mature fetus was 10.7 kilograms (23.5 pounds), and its length from crown to rump 63.5 centimeters (25 inches).

The one remaining fetus - an as-yet-unborn male pre-christened "Noah" (as in Ark) - is expected to achieve full neonatal development late next month. "We're most likely going to remove the animal by Caesarian, to increase its chances of a live birth," Damiani observed. "That would be the last week of November - just before Thanksgiving."

But will Noah be a 100 percent endangered-species gaur, or merely a chimeric hybrid sharing its surrogate mother's genes? Its pedigree, Damiani explained, "is more like 99 percent pure gaur, thanks to its mitochondrial inheritance."

He explained how he and his co-authors authenticated Noah's blood line:

"We took different tissues - heart, lung, stomach and so forth - from each of the three fetuses we analyzed. Then we subjected them to PCR, looking for bovine-specific and gaur-specific nuclear markers, to prove that the DNA derived from that fetus actually came from the gaur and not from the cow. We then went back and examined mitochondrial DNA, which is normally maternally derived. We expected that the mitochondrial DNA should have been bovine, since we used a cow egg.

"The mitochondrial analysis indicated that all the fetuses actually did have bovine mitochondrial DNA and not gaur mitochondrial DNA. But they all had gaur nuclear DNA. So Noah will be 99 percent gaur. That other 1 percent reflects the mitochondrial DNA that came from the cow."

But pedigree purity can become complete in the next generation.

"When Noah is able to breed," Damiani observed, "the concern is that he'll be passing all that 1 percent maternal DNA down to his offspring. That's not necessarily true, because the sperm mitochondria is inactivated by the egg cytoplasm. So if Noah mates with a normal gaur, wild or captive, his mitochondrial DNA will be all gaur. It will inactivate any of the bovine mitochondrial DNA that might be present in the sperm, which would then produce an animal that is 100 percent gaur - in both mitochondrial and nuclear DNA."

Where Will Noah Find Safe Harbor?

ACT has not yet decided on Noah's future. "We would prefer," Damiani said, "that he get placed in a zoological society, but there is also a chance we may have him located on a conservation facility, where he can spend his life in a natural habitat."

The team's next project is attempting to resurrect from its preserved tissue samples the Spanish ibex (Capra pyrenaica), or bucardo mountain goat, of which the last surviving specimen died early this year. Beyond that, no endangered pandas or extinct woolly mammoths need apply. As for the former, Damiani is already collecting eggs from the North American black bear (Ursus americanus), "which hopefully," he said, "can be applied to the panda (Ailuropoda melanoleuca)."

Woolly mammoths (Mammuthus primigenius) are definitely out of the running, Damiani concluded, "because you can't take this technology to Jurassic Park. The procedure requires intact DNA, which is not available in an animal that's been extinct for thousands of years."

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