LONDON ¿ A cloned sheep whose genome lacked one copy of the gene for the prion protein was born late last year in Scotland. The animal had to be put down 12 days after birth because of a heart defect, but its birth nevertheless represents the first example of a gene knockout in a mammalian species other than mice.

Many of the other ovine embryos that had been similarly manipulated so that they lacked a gene failed to implant. Many of those that did begin to develop died during fetal life. Of those that survived to term, four were stillborn and four live born, with three of those dying shortly after birth.

John Clark, joint head of the department of gene expression and development at the Roslin Institute in Roslin, Scotland, where the work was carried out, said that although the results were an ¿encouraging¿ step towards the use of the nuclear transfer technique to introduce precise genetic modifications into farm animal species for biotechnological applications, improving the efficiency of the methods used, to obtain more live animals, would require additional research.

¿This is the first time we have gotten close to deleting an important gene from a mammal other than the mouse,¿ said Clark. ¿In particular, this is a gene with tremendous implications for understanding diseases such as scrapie, bovine spongiform encephalopathy and new variant Creutzfeldt-Jacob disease.¿

The work is reported in Nature Biotechnology in a paper titled ¿Deletion of the alpha (1,3) galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep.¿ It was carried out at the Roslin Institute and funded by the UK¿s Biotechnology and Biological Sciences Research Council and Geron Corp., of Menlo Park, Calif.

To obtain knockout mice, scientists perform genetic manipulation on the genomes of embryonic stem cells, which are easy to grow in culture and can then be added back to a healthy blastocyst to form a genetic chimera. It is then a relatively simple matter to breed mice homozygous for the genetic alteration from the offspring of the chimera.

Unfortunately for researchers who want to carry out genetic manipulation in any other mammalian species, no one has yet isolated embryonic stem cells from any species other than the mouse, despite extensive efforts. Until recently, the only way that such species could be genetically modified was by simply injecting the extra genetic material into a recently fertilized egg. This method gives no control over where the injected DNA is incorporated in the genome, and it only allows genes to be added, not specifically knocked out.

To circumvent the problem, scientists at the Roslin Institute further developed the technique known as nuclear transfer, made famous by the birth of Dolly. This involves genetically modifying cultured cells before removing their nuclei and inserting these into enucleated eggs, which are then transferred to the uterus of a sheep.

This technique has been used by the Roslin Institute and PPL Therapeutics, also of Roslin, to create Polly the sheep and her sisters, which had extra genes for the human blood-clotting factor IX that was expressed in the mammary glands. As a result, the human protein was present in their milk. In this experiment, however, cells were simply transfected with the extra genetic material. Harry Griffin, assistant director of the Roslin Institute, explained that this technique gave no control over the site of insertion of the extra gene, which could appear in multiple copies.

Producing knockout sheep is more difficult, not least because both copies of the gene in question must be deleted. The Nature Biotechnology paper reports two separate attempts to delete genes.

The first was encoding the enzyme alpha(1,3)galactosyl transferase. This enzyme is present in all organisms except Old World monkeys, apes and humans. It catalyses production of the disaccharide galactose-alpha(1,3)-galactose, which may be important in mediating hyperacute rejection of transplanted organs. If sheep that lacked this enzyme could be obtained, researchers would be able to find out if their organs were less likely to be rejected. If they were, sheep may prove a suitable source of organs or tissues for transplantation into humans.

The second gene encoded the prion protein. In its modified form, the prion protein causes a range of transmissible spongiform encephalopathies, including scrapie, bovine spongiform encephalopathy and new variant Creutzfeldt-Jacob disease in humans. When this gene is knocked out in mice, the animals are resistant to scrapie.

¿If you could knock out both prion protein genes, then you would have an animal that could form the basis of a flock or herd from which you could get prion-free products for biomedical applications. Prion-free gelatine, for example, could be used in packaging drugs,¿ Griffin said.

He warned, however, that ¿substantial technical hurdles¿ remained to be overcome before researchers would be able to keep cells alive in culture long enough to carry out two rounds of gene knockout and selection, and still remain viable for nuclear transfer.

Clark said he and his colleagues will now be concentrating on trying to improve the efficiency of generating viable knockout livestock.

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