For those readers who feel a sense of déjà vu, you have seen the pig on the cover before. She is one of five piglets—named Noel, Angel, Star, Joy, and Mary in keeping with their December 25 birthday—that occupied news pages in early January. After delivering the pigs, PPL Therapeutics' subsidiary in Blacksburg, Virginia, also delivered a premature announcement, claiming the world's first report of cloned knockout pigs. In fact, another group of researchers collaborating with Immerge BioTherapeutics had produced a litter of four cloned knockout piglets months before, the results of which appeared in Science (295, 1089–1092, 2002) a couple of days after PPL's release.

Competition was fierce because these clones are the latest step in the race to turn pigs into organ factories for humans. The gene (GGTA1) that was deleted in these animals encodes α1,3-galactosyltransferase, which synthesizes one of the most important antigens in eliciting hyperacute rejection and (to a lesser extent) acute vascular rejection to xenografts. Importantly, because of the differences in immune responses to organs and cells, the lack of GGTA1 is likely to be particularly important for transplants of whole pig organs.

Essentially, two advances were required to produce the knockout piglets: adaptation of nuclear transfer technology to pigs (no mean feat considering the notorious fragility of pig embryos and the idiosyncrasies of pig reproduction); and the refinement of homologous recombination technology to enable specific targeting of genes implicated in immune rejection. Both groups went about creating their knockouts using a similar approach (despite differences in vectors, pig strain, and means of preparing sows for artificial impregnation). Gene targeting was used to inactivate GGTA1 in pig fetal fibroblast cultures, cells containing the deletion were then selected, and nuclear transfer was carried out to generate embryos that lacked one copy of the gene. The PPL paper, presented on page 251, provides independent confirmation of the results obtained by Prather and colleagues in Science, relates previously unreleased data confirming targeting of GGTA1 in pigs, and confirms deletion of the gene via Southern blots.

The next task for researchers is to produce—by either breeding or further rounds of targeting/cloning—pigs lacking both copies of GGTA1. When they've done that, they need to engineer pigs to carry five or six more genes that inhibit human complement activation and clotting around the xenograft, and then target pig adhesion molecules that could recruit human inflammatory cells. Put simply, we are still a very long way from ever turning this research into a clinical reality. What's more, the inability of current detection technologies to verify that transplants are free of viral contamination could condemn the field to regulatory oblivion, particularly if the European regulatory authorities continue their predilection for the precautionary principle and zero risk.

With advances in autologous stem cell technology and artificial organs gathering pace, xenotransplantation companies need to start making progress, and fast. The knockout cloning technology should enable a more systematic and rapid investigation of the antigenic targets involved in immune rejection. But will that be enough? Seven years ago, xenotransplantation pioneer Imutran (now out of business) predicted animal organs would be clinically available by 2002. If transgenic pig organs take another seven years to reach the clinic, stem cells and tissue engineering will be providing alternatives and the patience of those bankrolling xenotransplantation may have run out.