By David N. Leff

Dolly, meet Charlie and Georgie.

These two four-footed, twin male newborns are not sheep (Ovis aries) but cattle (Bos taurus). Nor were they cloned in Scotland, like Dolly, but in a laboratory at the University of Massachusetts (UMass)-Amherst.

The pair of calves were born, to surrogate mothers, last week on a cattle ranch in Texas. They went public in Boston Tuesday at the 24th annual three-day meeting of the International Embryo Transfer Society.

Developmental biologist Steven Stice reported to the conference on: "Cloning and transgenic technologies combined offer new products for the biomedical and agricultural industries." He is chief scientific officer of Advanced Cell Technology Inc. (ACT), in Worcester, Mass., and an adjunct professor at UMass.

Stice told the audience how he and his associate, developmental biologist James Robl, had cloned Charlie and Georgie, using procedures that differed markedly from technologies employed by the Scottish creators of Dolly at the Roslin Institute near Edinburgh. Robl, a full professor at UMass, is a cofounder and scientific consultant to ACT, and program chairman of the Embryo Transfer Society meeting.

Comparing the Roslin approach that created Dolly with ACT's methods, Stice told BioWorld Today, "We think there are at least five or six differences between what we do and what they do." He counted the ways:

"They use quiescent cells, that are not growing, to get closer to synchrony with the eggs they are putting into the cells. They then fuse them, and cause them to start developing right from the start.

"We don't do that. We use cells that are propagating, let them stay in the egg for a while — anywhere from two to eight hours — then start developing into an embryo at a little later stage."

Stice continued: "The eggs we use we collect by the thousand from slaughterhouse cows. We then mature selected ova to a stage where we can put them into the cloning process.

"Whereas they, at Roslin, go in and superovulate a ewe, get her to a certain stage, then take the eggs for cloning."

Another difference: "We culture our embryos in an incubator, then do a nonsurgical transfer into a surrogate cow. They are doing surgical transfers."

Stice went on to point out that unlike Roslin's herd of sheep, "We don't have a herd of cows here in Massachusetts. What we're doing is having the eggs shipped to us overnight by Federal Express from a Midwestern slaughterhouse. Then seven days later, after culture, we're shipping 100-to-200-cell embryos to an embryo-transfer company called Ultimate Genetics Inc., in Franklin, Texas, which puts them into cows the next day. It's a less expensive way of doing things."

Twins One Day, Triplets The Next

ACT's first two animals, Georgie and Charlie, serve as initial proof-of-principal models, Stice explained. Those twin male calves became triplets over the past weekend, with the birth of a third calf, as yet unnamed. "And a couple more are on the way," Stice said.

He cites as twin innovations in the cloning and genetic engineering technology that he and Robl have now demonstrated, "the first animal cloned from somatic cells in cattle, and the first transgenic in cattle."

The time elapsed from getting a fetus from the slaughterhouse, genetically modifying those cells, confirming that a cell has the gene of interest in it, then using those in the nuclear transfer procedure, takes on average three weeks. From there on, it's a matter of nine months' bovine gestation — 283 days for females to be born; 285 for males."

The next item on ACT's agenda is to raise up a herd of cows carrying the gene for human serum albumin expressed in their mammary glands, and recovered from their milk.

"I can't go into details on that, under our agreement with Genzyme," Stice said, "but we have started work on it, and that work is progressing." This is a collaborative project with Genzyme Transgenics Corp., of Framingham, Mass., announced in October last year.

Albumin is a high-value protein widely used in blood transfusions instead of whole blood, Stice explained. "It's the major component, which maintains the osmotic pressure of the blood vessels." He noted that a recent press release from Genzyme Transgenics described human serum albumin as a $1.5 billion market, with annual sales of dried plasma protein at 440 metric tons.

"An individual cloned, transgenic dairy cow," Stice pointed out, "is expected to produce approximately 80 kilograms of recombinant human albumin annually."

Over the past four years, UMass has applied for several patents covering the Stice-Robl cloning technology. "Our company started within the university," Stice said, "and has worldwide exclusive licenses to the inventions, with royalties, of course."

Next: Parkinson's, Pig Organ Grafts, Mad Cow Disease

Besides "pharming" females for useful proteins, ACT has several directly biomedical processes on its preclinical front burners. "With a collaborator at the University of Colorado," Stice recounted, "we've actually taken neural cells from cloned, transgenic cattle fetuses and transplanted them into a rat model of Parkinson's disease (PD). The results look quite promising."

He told the society meeting that for PD therapy "everybody would like to get away from transplanted human fetal cells, derived from abortions. Our cloned, transgenic cells would be an alternative source."

Another active project to which he alluded is "cloning transgenic, somatic cells in pigs. We haven't produced an offspring," he observed, "but we have produced embryos, with intent of working in the area of xenotransplanted organs." Given current concerns over retroviral infection from grafted porcine tissues, Stice made the point: "If there are only a couple of those viruses that are a problem in the pig, we should be able to use this technology to knock them out."

And applying the transgenic cloning to animal husbandry, he suggested "first off, mass-producing all female progeny for dairy farmers, and all-male offspring for beef producers."

On this note, Stice concluded, "The thought of knocking out the gene responsible for bovine spongiform encephalitis — mad cow disease — would be an interesting one as well, and would be conducive to this technology." *

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