By David N. Leff

Dolly the sheep is no longer the only kid on the block. The same Scottish scientists who cloned that celebrity ewe three years ago have now come up with three newly cloned lambs that see Dolly and raise her one.

Today's issue of Nature, dated June 29, 2000, reports the ovine blessed event in an article titled: "Production of gene-targeted sheep by nuclear transfer from cultured somatic cells." Its co-authors are at PPL Therapeutics Ltd., of Edinburgh, Scotland. The senior author is molecular embryologist Alexander Kind, head of cell biology at PPL.

"The three female lambs," PPL's managing director, Ron James, told BioWorld Today, "were produced from targeted fetal fibroblast cells, not adult cells, as we used with Dolly. But the method is essentially the same."

Two of the three lambs, pictured in Nature, born in July 1999, were named Cupid (god of love) and Diana (goddess of hunting and childbirth). "The reason why we called them Cupid and Diana," James explained, "had nothing to do with their sex, both being female, but had to do with the fact that the Cupid and Diana of mythology are associated with bows and arrows - and targeting. That was our thought behind it.

"The third lamb," he continued, "though just as healthy as the other two, never got formally named." The reason why, he pointed out "is that although the sheep can all be derived from exactly the same genetically modified cell, they're not necessarily all born at the same time - because you can go back to your cells and do another one. And we had two healthy sheep that were still alive after a week, which we named when we went public before the third one was actually born.

"The essence of what this Nature paper is about," James went on, "is the fact that the genetic modifications are targeted. For a long time, people have been able, for example, to delete genes in mice to make animal disease models and do various other things. Nobody until now has had the ability to make a precise genetic modification at a predetermined site in a livestock animal."

Targeting, Yes; Randomization, No

"We could never do that in the old way in which we used to make transgenic mice," James observed, "which was to poke a fine needle into the nucleus of a fertilized egg and inject some genes. When these inserted, which wasn't often, they inserted randomly.

"The whole concept behind the cloning of Dolly in the first place," he recalled, "was that it would give us a much better way of genetically modifying animals. So our next step after Dolly - who was not genetically modified at all - was to do a random insertion. Actually it was a Factor IX gene we used, and produced a sheep called Polly, at the very end of 1997.

"But that was a kind of random insertion of the transgene into the genome," James recounted. "What we have now done in these three lambs is very precisely locate the gene insertion - actually in the site that produces collagen. We chose that site because collagen is highly expressed in the mammalian body. It could have been any site you like."

The co-authors produced their first new-generation animal with just a neomycin resistance marker gene in that site. The second one got an additional gene that expressed alpha1-antitrypsin (AAT) in the same collagen site. "Incidentally," James observed, "it produced more AAT than we had ever seen from that gene in a randomly integrated site. That told us the collagen site is obviously a good one for protein expression. Not too surprising because collagen is about 1 percent of the messenger RNA that's produced by the body anyway."

As to the yield, James noted, "The 80 embryos transferred into a total of 42 final recipient mothers produced six lambs that survived more than a week and three that survived more than six months - now going on a full year. More important than the efficiency of the nuclear transfer," he added, "which is probably broadly comparable with what's been achieved before, is the efficiency with which the targeting event took place. In some places that was as high as 50 percent of the cells examined after selection. And that's very, very efficient, compared with what's been done before."

James observed, "This leads on to three potential outcomes:

¿ "One could now consider animal disease models in larger mammals by doing knockouts.

¿ "We can consider gene insertions for high production of therapeutic proteins - in our case in the milk, but wherever else you want to go. It's a question of choosing a site where you know the expression level is going to be commercially useful.

¿ "As for the third, PPL has an interest in xenografts - the idea of making pigs' donor organs compatible with human recipients. And we believe that the preferred solution to hyperacute graft rejection is to inactivate a particular gene that puts a particular sugar group - alpha-1,3- galactose - on the surface of the pig's cells, which makes them recognizable to the patient's immune system as foreign."

Porcine Donor Organ Rejection Rejection

"I think the next thing to do in this area," James said, "is try and put together the pig cloning technology with the gene targeting to make the knockout pig, as part of our xenotransplantation program. I think that would be the next milestone event for PPL".

The company already has cloned five pigs, as yet unpublished. When will that program come to fruition? "I have absolutely no idea," James confessed. "This is research. What I can tell you is that it won't be in the next several months, because pigs are pregnant for four months. It's going to take at least that long - probably longer."

Meanwhile, Dolly the dowager first-cloned sheep, "is living the life of a relative celebrity and experimental animal," James volunteered. "She's now had certainly two, probably three, lots of lambs. She's fit and healthy, and apparently not aging faster than she would normally be expected to, for a sheep born when she was.

"This has got to do with the fact that Dolly was made from a 6-year-old's cells," he continued, "which had shortened telomeres. But the evidence seems to be that one's got more than enough telomeres for at least one lifetime, and maybe two. So losing a few of those doesn't seem to be detrimental."