By David N. Leff
Science Editor
Dolly the sheep has become a household word as the first and only large mammal cloned from a single egg. Now meet Tommy, Andy, Timothy and Anthony, four bull calves born alive a year ago in Japan.
Dolly's egg donor was a nameless 6-year-old ewe in Scotland, now deceased. The cloned calves' cellular progenitor is a prize Japanese stud bull named Kamitakafuku. This genetically elite black cattle animal is 17 years old, and has sired nearly 160,000 bovine progeny. He is the proud property of Japan's Kogashima Prefectural Cattle Breeding Development Institute.
The father of the technology that produced these first-ever cloned calves is animal scientist Xiangzhong ("Jerry") Yang, who heads the University of Connecticut's Transgenic Animal Facility at its campus in Storrs. Yang is corresponding author of a paper to appear in an upcoming issue of the Proceedings of the National Academy of Sciences (PNAS), which was made public on Jan. 4. Its title: "Six normal cloned cattle produced from adult fibroblast cells after long-term culture."
For openers, the Japanese scientists scraped fibroblasts - somatic cells - from the ears of their star bull, and set up two long-term cultures using Yang's technique - one for two months of passaging, the other for three. They brought these cultures to Connecticut, and there prepared them for nuclear transfer. Then, back in Japan, they inserted them in enucleated, unfertilized eggs, which were placed in the wombs of surrogate mother cows.
Four of the six were born in December 1998, but two of those siblings succumbed perinatally to difficulties in labor unrelated to their cloning origin. The other two live births occurred in February 1999. Yang explained: "Long-term culture of somatic cells is essential for the possible targeted genetic manipulations of donor cells to create targeted genetically altered cells, tissues, organs and animals via cloning. Live clones have been obtained from adult somatic cells in sheep, mice and cows; however, these clones all came from donor cells after short in vitro culture, which did not allow future targeted gene manipulation."
Reproductive physiologist Michele Barber, a co-author of the PNAS paper, told BioWorld Today: "Culture long-term allowed for genetic manipulations of the DNA in these cells - for designer clones, if you will, that have genes either added, deleted or mutilated. Of course, this is great for biomedical research. I think that where our paper really shines is that we used long-term culture."
Somatic Cells Beat Pessimists
Thomas Chen, who directs the Biotechnology Center at Storrs, observed that, "Before this experiment, people always believed that somatic cells, being fully differentiated, are not totipotent. That is, they cannot be used to generate embryos. But Yang's result says, 'No, no, if you culture the cells under the appropriate conditions, then you can use them for the purposes of developing embryos.'" Those conditions included passaging these cultures 30 times before initiating the nuclear transfer.
"Since her birth three years ago," Chen went on, "Dolly has not been without difficulties. There is now a question concerning the age of cloned animals - the hypothesis that the length of the telomeres capping their chromosomes is a determining factor for the aging process. The genetic material she was copied from is aging at the rate of the older sheep from which she was cloned. And Dolly is not able to address that question very well for the reason that her donor ewe is already dead.
"In Yang's study," Chen pointed out, "all the animals used as a donor are still there, so a large number of the samples available are ready for him to address this aging question very thoroughly."
The American and Japanese co-authors of the new cloning technique foresee clear potential payoffs. "The first commercial application," Chen said, "is this: Traditional ways of making transgenic animals start by directly injecting genes into newly fertilized eggs. This works for mice, but not for larger mammals," he pointed out, "but in the case of the cow or pig and so on, the efficiency rate was less than one percent.
"In contrast," he continued, "with an embryo-cloning technique like ours, after you go through the gene transfer procedure, you have produced one transgenic animal in which you might be interested. Then, you can easily propagate that animal to produce a large number of identical copies.
"Any of the pharmaceutical proteins, which are now made easily by recombinant gene technology, can be produced far more efficiently by this cloning technique. For example, clotting Factor VIII is one of the proteins that, by recombinant DNA technology, is expressed in bacteria, yeast and so on, which are not practical vectors. Using this cloning approach, you transfer the Factor VIII gene into a cow producing milk. Then you isolate the transgenic animal that you want. All you need is one that you can immediately propagate into a large number of copies by Yang's method."
"The clotting factor is a good example, " Barber added, "because many other anticoagulation proteins are currently isolated from blood. So you can eliminate the use of scarce blood products, which is very positive due to infectious diseases."
Payoffs In Human Medicine, Agriculture
As for direct human medical applications, Chen observed, "In organ transplantation now, people are testing pig organs, but the problem there is histocompatibility - immune rejection. So using transgenic techniques, you can transfer genes into the pig, which will prevent this kind of problem. But," he added, "the success rate is very low, so to produce large numbers is difficult. By this cloning technique, all you need is one organ-producing pig, from which you can immediately clone a large number of duplicate animals. And a similar argument applies for antibody or vaccine aspects."
"We're working now on increasing the cloning efficiency of the process," Barber said, "because while the technique is worked out, it's still woefully inefficient. Also, making sure the health status of our cloned animals is normal. That's very important, especially looked at in any agricultural and medical sense. You certainly want these animals to lead a healthy life and be reproductive."