By David N. Leff

Steve Stice, who presented his firm's "cloned transgenic" technology to a Boston symposium earlier this week is co-author of an article on the subject in today's Science, dated May 22, 1998.

He granted "quite a bit" of textual homology between his talk and the Science paper, titled "Cloned transgenic calves produced from nonquiescent fetal fibroblasts."

"At the Massachusetts Biotechnology Council symposium," he observed, "we didn't go into detail on our reprogramming approach, which is the most striking new information in this paper."

Stice, chief scientific officer of Advanced Cell Technology Inc., in Shrewsbury, Mass., told BioWorld Today how this fibroblast reprogramming works. "We were able to take a fibroblast cell that is near death, close to senescence, put it through the nuclear transfer procedure again, then collect fetuses that had fibroblast cells with a normal lifespan. By going through the cloning process, we could turn back the clock on those fibroblasts so they behave like new cells."

Twenty-eight Embryos Yield Three Calves

"As we reported in Science," he continued, "we transferred 28 cloned transgenic embryos into 11 recipient cows, and got back three healthy calves. To do that with traditional transgenic technology would probably take several thousand embryos, and at least a couple of hundred recipient animals.

"With previous microinjection techniques," Stice pointed out, "about 500 embryos would have to be injected and transferred to recipient cows to get one transgenic offspring. For our nuclear transfer technique with transgenic somatic cells, the transfer of nine embryos to four cows produced one transgenic offspring. With this approach an entire herd of appropriate sex transgenic cattle could be produced in one generation.

"Fibroblast cells have a certain life span in culture," Stice narrated. "So they can undergo a certain amount of cell-doubling — the Hayflick limit — before they stop dividing and die. In our cell line, when we isolate them from a fetus, they can do about 30 cell doublings in a Petri dish.

"We let one cell line go out to the end, one cell-doubling away from being in senescence. Then we took those cells at the end of their 30-doubling life span, put them into the nuclear transfer procedure, and produced an embryo from that end-stage fibroblast cell.

"From that embryo," Stice went on, "we produced a fetus and collected fibroblast cells from it. It's sort of a circular situation, where those new fibroblast cells in fact have another 30 cell doublings before they stop dividing.

"This has an interesting impact," he pointed out. "We could first do genetic modifications in the first round of the fibroblast's life span. And then make further genetic modifications — add or subtract other genes — and go through the process again to make the actively dividing reprogrammed fibroblast cells renew their life span. In fact, if we continually did this, we would have a limitless life span for those fibroblasts."

The researchers then inserted two transgenes — one encoding beta galactosidase, the other neomycin resistance — as markers into the fibroblasts, and fused those now-transgenic cells to mature bovine oocytes (eggs) from which the nuclei had been removed.

After seven days of priming in cell culture, the resultant embryos were implanted nonsurgically into the uteri of recipient cows. About nine months later — normal gestation period for bovines as for humans — George and Charlie were born.

Stice and James Robl, professor of reproductive and developmental biology at University of Massachusetts, are co-inventors on several pending patents in the U.S. and abroad. "They protect the techniques and procedures we have used," Stice said, "to produce transgenic and cloned animals. Also the area of embryonic stem cells.

"The area that we would commercialize first," he added, "will be for high-value proteins in milk. That will certainly take some time to go through clinical trials. On the agricultural side, though, "we will be producing clones next year for very niche and specific applications, such as copies of superior genetic animals for breeding purposes." *