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This little piggy goes to market, this little piggy is for transplants
By Bijal P. Trivedi

Two teams, one in Japan and one in the United States, are publishing independent reports on pig cloning in competing journals. The Japanese cloned a pig from fetal skin cells, using a technique almost identical to one used to clone mice two years ago. Their piglet, a black Chinese Meishan called Xena, was born on July 2, 2000. The work is reported in the August 18 issue of Science. The US team of PPL Therapeutics announced they had produced five cloned piglets—Millie, Christa, Alexis, Carrel, and Dotcom—on March 14 of this year, but failed to supply any details of the method used. Their report will be published in the August 24 issue of Nature.

Xena, the cloned pig

Researchers believe cloning is an important step toward creating genetically modified pigs whose organs can be used for transplantation into humans. Without genetic alterations to change the surface sugar molecules and proteins on pig cells, transplanted organs are instantly rejected by the human body. Once an animal with these changes exists, the expectation is that cloning will allow identical animals to be mass-produced, providing a cheap and reliable source of safe organs. It also holds promise for the meat industry, which could create replicas of the best bred and tastiest livestock.

Cloning is based on the idea that because every cell in the body contains the same genetic package—the same type and number of chromosomes with all the same genes—it should be possible to replicate an animal using the genetic information in any cell. In an adult cell many of the genes are turned off; removing just the nucleus from a body cell and placing it in an egg somehow allows the genes to return to their embryonic state, says Tony Perry, of The Rockefeller University and an author on the Science paper.

But taking a nucleus from a skin cell, for example, transferring it into an enucleated egg, whose own genetic material has been removed, and placing it back into a surrogate mother for development has been particularly tricky in pigs. The two teams have developed slightly different approaches.

Technique used by the Japanese team
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The Japanese team removed the nucleus of a fetal skin cell from a black Meishan pig and injected it into an enucleated egg cell from a white Landrace pig. The 'white' egg containing the 'black' nucleus recovered in a broth for a few hours and was then given an electrical pulse that triggered the egg to begin dividing and forming an embryo. The researchers then took the 8-celled embryo and implanted it into a surrogate mother pig. 110 of these embryos were distributed between four surrogate mothers. Xena was the only clone born and was delivered naturally.

The PLL team, in Blacksburg, Virginia, used a different technique that involved transferring the nucleus twice. PPL took an entire nurse cell from an adult sow and fused it with an enucleated egg. The fused cell was then given a short electric pulse. "This first nuclear transfer is the reprogramming step for the genetic material," says Dave Ayares, of PPL. The nucleus was then transferred to a naturally fertilized pig egg from which the sperm and egg DNA had been removed. "We think that the second transfer of the nucleus into a naturally fertilized egg is what makes this technique efficient," says Ayares. After the second transfer the cells were implanted in surrogate mothers. View an image of this technique.

Fear of animal viruses cast doubt on use of pig organs

PPL completed 72 double nucleus transfers that produced five clones; three were identical clones from one nurse cell, the other two were identical clones from another. The piglets were all delivered by Cesarean section and weighed around 2.72 lbs, about 25 percent less than piglets from a natural mating.

PPL intends to use the technology to clone pigs lacking the gene for an enzyme that adds a sugar to the surface of pig cells. The sugar is thought to be responsible for the "hyper-acute rejection response" in humans, which destroys the transplanted sugar-coated pig organs, turning them black and necrotic within three minutes.

"We have pig cells with the gene knocked out, and we've done cloning, so now we just need to throw the technologies together," says Ayares. Humans have multiple pathways for rejecting foreign organs, and at least five other modifications will need to be made to the pig cells before the organs would be "humanized enough" to avoid rejection. PPL expects to have an organ suitable for human trials by 2005, according to Ayares.

The expectation of using pig organs for transplants is dampened by a report, also in the August 24 issue of Nature, that all pig cells harbor porcine endogenous retroviruses (PERVs), which can infect human cells in the test tube. The researchers also showed that insulin-producing pig cells transplanted into diabetic mice could migrate beyond the region of the transplant and spread viruses throughout the body. "PERV doesn't cause disease in mice, the mice aren't even sick, and we had to bend over backward to actually detect these viruses. PERV is just not very scary, but the risk of it infecting human cells, though small, is real," says Daniel Salomon who led the research at The Scripps Research Institute in La Jolla, California.

"Salomon's report concerns everyone in the xeno-transplant arena," says Ayares. However, a study published in Science last year found no evidence of PERV infection in any of the 160 people who had received some type of pig transplant.

Another blow came when researchers at the Roslin Institute in Edinburgh, UK, which became famous after creating Dolly, the first cloned sheep, announced it would reduce research on organ modification in pigs. Initially citing a fear of pig viruses spreading to people, the institute later stated that the decision had been made for commercial reasons.

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Polejaeva, I.A. et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 406, 505-509 (August 24, 2000).
van der Laan, L.J.W. et al. Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature 406, 501-504 (August 24, 2000).
Onishi, A. et al. Pig cloning by microinjection of fetal fibroblast nuclei. Science 289, 1188-1190 (August 18, 2000).

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