LONDON ¿ When mammals are cloned by transferring the nuclei of adult somatic cells into oocytes from which the nucleus has been removed, the death rate during late pregnancy and the first few days of life is very high. Depending on the experiment, up to three-quarters of these animals fail to survive, although the reason for this high mortality is not known.

Now, a team of French researchers have reported that a calf they cloned in this way died as a result of atrophy of the thymus, together with an inability to regenerate blood cells, after more than six weeks of apparently normal development. At the time the animal was born, in July last year, it was the first successful example of bovine nuclear transfer using skin cells as a source of nuclei. The donor animal was also a clone.

The report appears in the May 1 issue of The Lancet, in a paper titled ¿Lymphoid hypoplasia and somatic cloning.¿

Xavier Vignon, a researcher at the Institut National de la Recherche Agronomique in Jouy en Josas, France, and last author of the paper, told BioWorld International: ¿This example shows that, in the case where adult somatic cells are used as a source of nuclei, there are occasionally long-term effects associated with reprogramming of the nucleus.¿ Vignon and his colleagues conclude that this observation should be taken into account in debates on reproductive cloning in human beings.

Alan Colman, research director of PPL Therapeutics, of Roslin, Scotland, commenting on the report, said he did not believe it revealed anything new because it is already widely recognized that postnatal mortality following the nuclear transfer technique is high. Animals produced in this way at PPL had occasionally died, but poor development of the thymus had not been the cause. He added: ¿It should also be noted that the animal resulted from the cloning of a clone and this seems an unnecessary complication, given the newness of the technology.¿

Colman said a research group in New Zealand recently reported that it had obtained ten cloned and healthy calves using cells taken from a single adult cow of an endangered breed. He said: ¿The French results should be viewed in this context, which shows that the technique can work quite well. Nevertheless, many of the embryos produced by the technique do not develop properly, indicating that there is much yet to learn before somatic cell cloning becomes efficient.¿

In the French study, the animal (which later died) was cloned as follows. First, nuclei were obtained from a sample of epithelial cells taken from the ear of a calf. This donor had itself been cloned, but the source of the nuclei for this animal was embryonic cells. The donor nuclei were then placed in enucleated oocytes. Out of 175 nuclear transfer embryos, six blastocysts were obtained. These were transferred to five recipient foster mothers. One of these cows became pregnant, and gave birth to a calf. Tests showed that the genotype of this animal was the same as that of the animal that donated the nuclei.

One year earlier, Vignon explained, a calf obtained by nuclear transfer, where the source of the nuclei was fetal muscle cells, had died after only seven weeks. The animal had suffered an accidental injury but had developed an overwhelming infection, which proved fatal despite intense treatment with antibiotics. Tests showed that it had an underdeveloped thymus gland, a condition called thymic hypoplasia.

With the calf born last year, therefore, regular blood samples were taken every other day. Levels of lymphocytes and erythrocytes were normal at first. One month after birth, however, lymphocyte levels fell rapidly; 40 days after birth hemoglobin levels also fell. The calf died at the age of seven weeks from severe anemia. Attempts to correct the anemia with blood transfusions and iron supplements had no effect.

On necropsy, the thymus, spleen and lymph nodes were all found to be much smaller than expected, and the team concluded that the hypoplasia had probably been present at birth. Tests on stored blood samples showed no clear evidence of immunoglobulin G synthesis.

Renard and his colleagues discuss in their Lancet paper what the possible causes of this defect could be. A lethal defective genotype in the donor animal can be ruled out, they say, because the donor animal belongs to a group of three other identical animals which are currently healthy and now more than 18 months old. Similarly, the culture conditions and nuclear transfer technique are not likely to be to blame, because the same methods were used to produce the donor animal.

Instead, they conclude, the cause of death is likely to be ¿factors inherent to somatic cloning ¿ i.e., the random choice of a cultured donor cell that bears a mutation ¿ or the lack of fine-tuned regulation during somatic reprogramming.¿

The group is now pursuing further studies into the process of nuclear reprogramming, which has to take place when the nucleus of a differentiated cell is placed into an enucleated oocyte. Vignon told BioWorld International: ¿Nobody knows exactly what happens at the molecular level in the nucleus when you put it in the new environment of the enucleated oocyte. We need to understand in order to improve the efficiency of the reprogramming process.¿

He added: ¿We are also stepping up our investigations on these animals, both during pregnancy and after birth, in terms of blood monitoring, echocardiography and ultrasonographic examination to look at organ development, including that of the thymus. We will also be performing analysis of circulating factors such as growth factors to see if we can identify one common syndrome in the cloned animal.¿ n