Serial nuclear transplantation offered a means of overcoming both difficulties. A sample of nuclear-transplant embryos whose development was so abnormal they would have died before reaching the muscular-response stage provided nuclei for serial-transplant clones. In 70 percent of the serial-transplant clones some of the embryos developed as far as the muscular-response stage or beyond it. By adding the proportion of nuclei shown by first transplants to be able to support muscular-response differentiation to the proportion shown by serial transplantation to possess this capacity, we can conclude that at least 20 percent of the intestine epithelium cells must have retained the genes necessary for muscle-cell and nerve-cell differentiation.
There is no reason to believe that muscle-cell or nerve-cell genes have been lost or permanently inactivated in the remaining 80 percent of transplanted intestine nuclei. There are many reasons why it might not have been possible to demonstrate their presence. For example, about 50 percent of all the eggs that received intestine nuclei failed to divide even once. When a sample of these eggs was sectioned, they were found to contain either no nucleus at all or else a nucleus that was still inside an intact intestine cell. In the first instance the nucleus presumably stuck to the injection pipette and was never deposited in the egg; in the second, the donor cell was never broken so as to liberate its nucleus, a technical error that is easy to make with very small cells. In both cases the developmental capacity of the intestine nuclei was not tested, and the recipient eggs that failed to divide should not be counted in the results.
It is clear from these experiments that the loss or permanent inactivation of genes does not necessarily accompany the normal differentiation of animal cells. This conclusion is not inconsistent with a recent finding: the "amplification" of the genes responsible for synthesizing the ribonucleic acid (RNA) of the sub cellular particles called ribosomes. This phenomenon was demonstrated in amphibian oocytes, the cells that give rise to mature eggs. The nuclear-transfer experiments just described do not exclude such amplification, which simply alters the number of copies of one kind of gene in a nucleus. Instead they show that specialized cells always have at least one copy of every different gene.
The inability of some transplanted nuclei to support normal development has attracted considerable interest be cause it is always found that the proportion of nuclei showing a restricted developmental capacity increases as the cells from which they are taken become differentiated. Furthermore, serial nuclear-transplant experiments conducted by Briggs and King (and subsequently by others) have shown that all the embryos in a clone derived from one original nuclear transplant often suffer from the same abnormality, whereas the embryos in a clone derived from another original transplant may suffer from a different abnormality. Some of the abnormalities of nuclear-transplant embryos can therefore be attributed to nuclear changes that can be inherited.
The discovery that these changes arise as a result of nuclear transplantation, and not in the course of normal cell differentiation, was an important one. This was first established by Marie A. DiBerardino of the Institute for Cancer Research, who made a detailed analysis of the number and shape of chromosomes in nuclear-transplant embryos. Abnormal embryos were usually found to suffer from chromosome abnormalities that were not present in the donor embryos, a finding that at once explains why the factors causing many of the developmental abnormalities of nuclear-transplant embryos are inherited. The fact that chromosome abnormalities arise after nuclear transplantation does not necessarily mean that they are of no interest; there could be a connection between the kind of chromosome abnormality encountered and the cell type of the donor nucleus concerned. In spite of an intensive search, however, no such relationship has yet been found.