We can now return to the question of whether or not genes are lost in the course of normal cell differentiation. Nuclear-transfer experiments are performed to answer this question on the assumption that if the combination of egg cytoplasm with a transplanted nucleus can develop into a normal embryo possessing all cell types, then the transplanted nucleus cannot have lost the genes essential for pathways of cell differentiation other than its own. For example, if a normal embryo containing a specialized cell type such as blood cells can be obtained by transplanting an intestine-cell nucleus into an enucleated egg, then the genes responsible for the synthesis of hemoglobin cannot have been lost from the intestine-cell nucleus in the course of cell differentiation. The only assumption here is that a gene, once lost, cannot be regained in the course of a few cell generations. It happens that the best evidence for the retention of genes in fully differentiated cells comes from two series of experiments carried out at Oxford on eggs of the frog Xenopus.
The fully differentiated cells used for these experiments were taken from the epithelial layer of the intestine of mutant tadpoles that had begun to feed. Intestine epithelium cells have a "brush border," a structure that is present only in cells specialized for absorption and that is assumed to have arisen as a result of the activity of certain intestine-cell genes. Not all the cells of the intestine are epithelial, but when the epithelial cells are dissociated, they can be distinguished from the other cell types by their large content of yolk, by the ease with which they dissociate in a medium that contains Versene and sometimes by their retention of the brush border.
The first experiments with intestinecell nuclei were designed to show that at least some of these nuclei possess all the genes necessary for the differentiation of all cell types, and therefore that some of the transplant embryos derived from intestine nuclei could be reared into normal adult frogs. Both male and female adult frogs, fertile and normal in every respect, have in fact been obtained from transplanted intestine nuclei. Although only about 1.5 percent of the eggs with transplanted intestine nuclei developed into adult frogs, all of these frogs carried the mutant nuclear marker in their cells; their existence therefore proves that at least some intestine cells possess as many different kinds of nuclear genes as are present in a fertilized egg.
Subsequent experiments with intestine nuclei were designed to show that many of these nuclei have retained genes required for the differentiation of at least some quite different cell types. In these experiments the criterion for gene retention was the differentiation of functional muscle and nerve cells by nuclei whose mitotic ancestors had already promoted the differentiation of intestine cells. Functional muscle and nerve cells are present in any nuclear-transplant embryo that shows the small twitching movements, or muscular responses, characteristic of developing tadpoles just before they swim. Out of several hundred intestine nuclear transfers, about 2.5 percent of the injected eggs developed as far as the muscular-response stage or further. The reason why the remainder did not reach this stage is not necessarily because that proportion of intestine nuclei lack the necessary genes. In some cases it is known to be the inability of certain recipient eggs to withstand injection; in others it is the incomplete replication of some of the transplanted nuclei or their daughter nuclei during cleavage. In either case a nuclear-transplant embryo should contain some cells with normal nuclei as well as some abnormal cells responsible for the early death of the embryo.