Dr. Takumi Takeuchi, from Cornell University, New York, USA told a media briefing that he and Dr. Gianpiero Palermo's team had compared imprinting abnormalities (the process where specific genes inherited from both parents are silent) in mice embryos derived from assisted reproduction techniques and from cloning.

We found significantly impaired development in the cloned embryos compared with those derived from more conventional ART techniques, said Dr. Takeuchi, and this has made us more convinced that reproductive cloning is unsafe and should not be applied to humans.

Drs. Takeuchi and Palermo were prompted to undertake the research by concerns about the increased incidence of imprinting abnormalities in children born after ARTs. The most prominent of these is Beckwith-Wiedemann syndrome, where children are born larger than normal.

Scientists also knew that cloned animals had been born with a similar condition, called large offspring syndrome ™. Dr.Takeuchi ™s team set out to study whether the disorders arising in the ART system and those in cloned animals were comparable. The team took mouse oocytes and divided them into three groups.   68 were inseminated by ICSI, 37 activated parthenogenetically (without involving male gametes), and 77 were cloned by injected a cell nucleus into an egg where the nucleus had been removed. Of this latter group, 43 underwent first embryonic cleavage (the first few divisions of an embryonic egg) and 15 became full blastocysts.

The embryos created by parthenogenesis and those from ICSI reached the blastocyst stage at the same rate, unlike the clones, where only 30% got that far, said Dr. Takeuchi. This appears to be due to the abnormal gene expression we saw in the cloned group. This not only explains the developmental impairment of the cloned group, but may in future be helpful in identifying environmental culture condition that are deleterious to the development of ART embryos, he said.

Dr. Takeuchi said that as yet it was difficult to make a direct link with a specific cause for the abnormalities. But there are a number of possibilities, he said. They could be linked to fertility medications utilized to induce superovulation, or the progesterone employed to help implantation; in vitro culture conditions which could be related to the length of the culture or the concentrations of certain media components such as serum or even a specific amino acid. Finally, we cannot exclude the contribution of the peculiar genetic makeup of patients ™ gametes, together with the specific ART procedure, he said.

Identification of gene expression abnormalities would help to monitor the development of reproductive techniques prior to their application to routine medical practice, said Dr. Takeuchi.

eshre/

They were able to mimic the spontaneous genetic mutation Connecticut researchers found in the fruit fly by feeding C. elegans specially engineered bacteria that knock down the activity of Indy. Their model netted a 15-20 percent increase in lifespan in addition to the other benefits. Unlike true genetic knockouts, with scientists completely removing both copies of a gene so 100 percent of function is gone or taking out one copy so the gene functions at half capacity, the MCG scientists cannot determine the exact gene activity level in their animal model. "These worms reflect what happens with reduced activity in the transporter," Dr. Ganapathy says. "But we don ™t yet have a stable mutant line. That is one of the aims for the NIH grant."

Oddly, the maximum benefit, at least in the fruit fly, doesn ™t come from zero activity. Rather flies live the longest with about half the normal gene activity. Dr. Fei wants to find the optimal degree of activity. He and his co-investigator, Dr. Ganapathy, already are working on a knockout mouse that has half the normal Indy activity so they can look at the impact on longevity in mice that usually live two years instead of a few weeks.

To confirm that the gene functions similarly in worms and humans, they also plan to take the Indy gene out of the C. elegans and replace it with the human gene to see if that reverses the effect. "We call it humanizing the worm," Dr. Ganapathy says.

He noted an interesting difference between worm and human genes is that the human Indy gene is more adept at transporting tricarboxylates or citrates, a primary precursor for fat and cholesterol. "If you find a drug which can block the function of this transporter, it might interfere with the use of citrate for fat and cholesterol synthesis which should help people lose weight and reduce their cholesterol," Dr. Ganapathy says.

Drs. Fei and Ganapathy also are working to identify compounds that can control gene activity. They may have to look no further than store shelves to find a good starting point: hydroxycitrate, an analogue of citrate found in the skin of the Indian fruit garcinia, already is being touted for its weight-loss and cholesterol-reducing properties. "We think the mechanism for how this compound works is at least partly by manipulating this transport system, " Dr. Ganapathy says, adding that studies of hydroxycitrate might point toward more specific, potent compounds.

The potential benefit derived from manipulating the activity of Indy has prompted the MCG Office of Technology Transfer and Economic Development to seek national and international patents on the transporter technology.

mcg