New transgene delivery technique takes a traditional route

An Italian-Australian research partnership has demonstrated a potentially revolutionary technique for producing transgenic animals, using nature's own time-tested vector for delivering genes to eggs: sperm.

Their achievement vindicates Italian researcher Prof Martaluisa Lavitrano, of the University of Milan-Bicocca, who announced in 1989 that she had succeeded in producing transgenic embryos by fertilising mammalian ova with sperm carrying transgenes.

Other laboratories were subsequently unable to reproduce her results. The combination of conditions that had allowed the sperm to take up and integrate new genes in their chromosomes remained elusive for a decade.

But as molecular geneticists saw for themselves at the Transgenic Animal Research Conference in Lake Tahoe, California yesterday, it wasn't just huff and puff.

Lavitrano's team, working with Prof Ian McKenzie's research group at Melbourne's Austin Research Institute (ARI), have blown away the doubters -- and their peers -- by using sperm-mediated gene transfer (SMGT) to create 18 transgenic piglets.

Nature reported last week that the Melbourne-Milan team had inseminated sows with sperm carrying transgenes encoding red, green and blue fluorescent proteins. Of the 100 embryos they recovered, 90 expressed at least one of the transgenes.

In Bologna, on April 21, two sows re-implanted with the transgenic embryos gave birth to two litters, totalling 18 piglets. Four piglets expressed a single fluorescent protein, seven had two proteins, and the remaining seven expressed all three -- glowing testimony to the technique's simplicity and efficiency for developing transgenic animals with multiple transgenes.

It was the prospect of using the technique to reliably introduce multiple genes into pigs that led McKenzie's team to collaborate with Lavitrano's Milan group. The ARI researchers are trying to develop humanised pig organs for human transplant patients, to remedy the chronic shortage of human donor organs.

McKenzie's team had had limited success using the notoriously inefficient technique of transferring genetically modified cell nuclei into enucleated eggs. The alternative route, microinjecting DNA into embryonic stem cells, has a success rate of less than 1 per cent in most animals, and doesn't work at all in pigs.

But Lavitrano's new SMGT technique worked beautifully. "The main attraction is its simplicity and speed, and low cost," McKenzie said. "For our xenotransplantation work, we're looking at putting in six genes."

Because the genes integrate in different chromosomes, they would segregate independently in the transgenic piglets' progeny. McKenzie says that once his team has the right combination of genes in place, it will be maintained by cloning the animal.

The pig organs will be humanised to prevent organ-transplant patients rejecting them, by adding new human genes and knocking out others. McKenzie's team has already successfully knocked out a pig gene called Gal, encoding an enzyme that attaches sugar molecules to proteins.

The ARI team described and patented the gene six years ago. It triggers a hostile immune reaction that can initiate rejection of the organ within 10 minutes.

McKenzie praised Lavitrano for her discovery. "There's no question she invented the techniques, but they couldn't be reproduced because they involved a lot of art.

"Our contribution was to use her techniques to introduce multiple genes.

McKenzie says the Austin Research Institute's company Xenotrans recognised Lavitrano as the inventor of the patents, even though the problem of reproducing her results prevented her patenting the original methodology.

But there are still some doubters, according to the Nature article.

It quotes Tom Newberry of Genzyme Transgenics Corporation, in Framingham, Massachusetts, as saying that, despite their inefficiency, nuclear transfer and microinjection may be commercially more attractive to large companies developing transgenic animals despite their inefficiency, because they do not require the special laboratory conditions of SMGT.