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Human genes in pig sperm for organ donors

MILAN, Italy, Oct. 21 (UPI) -- Italian scientists said Monday by piggybacking human genes in pig sperm they have created swine that someday might help serve as life-saving donors for organ transplants to humans.

These new pigs have tissues better able to resist the human body's immune rejection system. The research team hopes this will help improve "xenotransplantation," or organ transfer across species.

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"In the U.S., every 18 minutes a person dies on the waiting list for organ transplants without receiving one. Every 18 minutes is a lot. Xenotransplation could really be a solution," lead researcher Marialuisa Lavitrano, an immunologist and pathologist at the University of Milan-Bicocca, told United Press International.

This sperm-based technique also is relatively cheap and about 14 to 114 times more effective at implanting genes in pigs than the direct injection of DNA, the most common way of making genetically engineered animals.

If this technique works in other animals, such as cows, "there is a long list of potential applications this could be used for," said U.S. Department of Agriculture physiologist Bob Wall at the Animal and Natural Resources Institute in Beltsville, Md.

"You might want to make 'nutraceuticals' in the milk of farm animals to improve it for consumption or you might want to introduce genes that allow them to reduce a particularly devastating disease. You might want to change their metabolism to improve or reduce the amount of fat on them," Wall said.

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For the most part, scientists genetically modify animals by injecting DNA into eggs right after they are fertilized, a tricky and expensive operation. While this "microinjection" technique is fairly successful for mice, "it's about 10 times less effective for livestock than mice," Wall said. He suspects one reason is because scientists have bred lab mice to produce hardier eggs on average than livestock.

Since sperm are designed to deliver their DNA into eggs, Lavitrano and colleagues tried using sperm as gene carriers. In 1989, they reported sperm could absorb foreign DNA into specific places in their chromosomes. Other groups were, however, claimed they were unable to reproduce the results.

"They were skeptical. They thought it was too good to be true," she said.

Over the past decade, groups in Europe, China, Israel, Cuba and the United States were able to use sperm to implant genes into the eggs of sea urchins, chickens, frogs, honeybees and sheep. However, the foreign genes did not function.

After much tinkering, Lavitrano said her team has now optimized the technique. "We've discovered what to do -- how much DNA to use, when to give it, for how long, and in what condition," she explained. "We do not need any of the expensive equipment, microscopes or anything, that you need with microinjection."

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Instead of extracting sperm cells from pigs or using frozen sperm, Lavitrano's team coaxed two male pigs with healthy sperm to ejaculate. Such sperm are more mature, she said, and frozen sperm cell membranes are damaged. Unlike sperm extraction techniques, where pigs are then killed, Lavitrano added "we can use ejaculate for a long time."

The semen fluid was washed off the sperm, since it protects them from absorbing DNA. The bottles of cleaned sperm then simply had DNA mixed with them for two hours, with scientists flipping the flasks upside-down every 20 minutes to keep the sperm from settling.

The scientists used the gene for human decay accelerating factor, or hDAF, a protein found on cell surfaces that protects cells from their body's at-times misguided immune system. When eggs were fertilized with these modified sperm in vitro and implanted into mothers, 57 percent of the 93 piglets born had hDAF in their hearts, lungs, kidneys, ears and tails. DNA injection would have only led to a 0.5 to 4 percent success rate, Lavitrano said.

When cells from both normal and the modified pigs were exposed to human antibodies, normal pig cells survived less than 10 percent of the time, dying in about 30 minutes, while the modified pig cells usually survived nearly 100 percent of the time.

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Scientists want to program donated organs to make them appear friendly to the body. When a person normally receives an organ transplant, his or her immune system attacks it as if it were a foreign invader. Drugs that suppress the immune system can somewhat help, but carry the risk of weakening the body against genuine threats.

Since donated human organs are short in supply, scientists would like to turn to pigs. The organs of human relatives, such as chimps or monkeys, also are possibilities but swine grow and reproduce more quickly than primates, giving birth to larger litters with organs comparable to humans in size and anatomy, Lavitrano said. Since pigs and humans are less related than humans are to other primates, there also is less chance some germ will hop species to infect people, she added.

"Of course there are still a lot of ethical concerns with xenotransplantation," Lavitrano said. "The FDA, even the Vatican, has discussed the feasibility of this."

The hDAF gene only allows transplanted organs to escape the first phase of immune rejection, Lavitrano cautioned. "We need to add more genes to the pig genome in order to control the rejection reaction," she said. "We are not even close. This is not in the near future."

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Still, Lavitrano feels this represents an important first step. "I think the results are pretty damn astounding," Wall said. "This is now a reachable technique for a lot of folks."

The scientists described their findings this week in the Proceedings of the National Academy of Sciences.

(Reported by Charles Choi, UPI Science News, in New York.)

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