First came sheep, mice, goats, and cows. Now, two independent research teams have added pigs to the list of species that have been cloned. The results had been awaited eagerly, because pigs' physiological resemblance to humans makes them a potential source of donor organs.
Animal scientists have had trouble cloning pigs; techniques used in other livestock didn't work as well. Nevertheless, a Scottish company called PPL Therapeutics announced the birth of the world's first cloned piglets in a March press release. At the time, the company attributed its success to a new approach but wouldn't reveal any details. Meanwhile, Akira Onishi, an animal breeder at Japan's National Institute of Animal Industry in Tsukuba, and his colleagues were working out their own approach. This yielded a piglet, Xena, on 2 July; the results will be published in the 18 August issue of Science. (Click here for a video clip featuring Xena.)
Onishi and his colleagues used the technique that produced Dolly, the sheep that set off the cloning frenzy in 1997, but with some variations. To make Dolly, researchers took the nucleus out of a mature egg and replaced it with the nucleus from a cell of the ewe they wanted to clone; then they activated development with electrical pulses and implanted the embryo into a surrogate mother ewe.
As the source of their nucleus, the Japanese team took connective tissue cells from 24-day-old fetuses of a black breed of pig. As was done with Dolly, they first deprived those cells of nutrients to shut down cell division and most gene activity. But the next step--transferring the nucleus--was different; rather than fusing the donor cell with the enucleated egg cell, the team removed the donor nucleus with a very fine needle and injected it into the donor egg. This "microinjection" approach was pioneered by a team in Hawaii to produce Cumulina, the first cloned mouse (ScienceNOW, 22 July 1998).
Xena was the sole survivor of 110 cloned embryos placed into four surrogate sows. Her color easily revealed her genetic identity--although born to a white sow, she was black--and independent DNA analyses confirmed that her DNA matched the donor fetal cell. "It's a great success," says Philip Damiani, a reproductive physiologist at Advanced Cell Technology in Worcester, Massachusetts. "It proves that microinjection can be used in large animals and livestock."
The PPL team used the traditional fusion technique to marry the donor cell nucleus to the egg. But PPL's Irina Polejaeva then added a second nuclear transfer to the cloning protocol: As soon as the transferred nucleus expanded, as it typically does, the PPL team removed that nucleus once again and placed it in another egg cell that had just been fertilized. Although the researchers had first removed the DNA of both the egg and the fertilizing sperm, the egg was still primed for cell division and development, increasing the chances of success, the team says. Five piglet clones were born from some 401 embryos implanted into surrogate sows. The PPL findings still haven't been published, but Nature has posted a paper describing them on its Web site.
The jury is still out on which technique works best. But colleagues say both teams probably boosted their chances by taking mature eggs straight from a female pig before taking out their nuclei, rather than collecting eggs from slaughtered animals and maturing them artificially in the lab, which is what most would-be cloners were doing. And most likely, Damiani adds, "there will be more pigs on the way."