The aim of a nuclear-transplant experiment is to insert the nucleus of a specialized cell into an unfertilized egg whose nucleus has been removed. Ingenious attempts in this direction were made many years ago by constricting an egg just after fertilization and then letting one of the early-division nuclei that appeared in the nucleated half of the egg enter the non-nucleated half. This method, however, is applicable only to the nuclei of early embryos whose cells are not normally regarded as being specialized. The first real success in transplanting living cell nuclei into animal eggs was achieved in 1952 by Robert W. Briggs and Thomas J. King, both of whom were working at the Institute for Cancer Research in Philadelphia. Their method, which has been generally adopted in subsequent work, involves three steps. Owing to the fortunate circumstance that the unfertilized egg of an amphibian has its nucleus (in the form of chromosomes) located just under the surface of the egg at a point visible through the microscope, it is not difficult to obtain an egg with no nucleus. This can be done by removing the region of the egg that contains chromosomes with a needle or by killing the nuclear material with ultraviolet radiation. The second step is to dissociate a tissue into separate cells, each of which can be used to provide a donor nucleus for transplantation. The cells separate from one another in a medium lacking calcium and magnesium ions, which are removed from the embryo more quickly by adding to the medium a chelating substance such as Versene.
The third and most difficult stage in the procedure involves the insertion of the donor-cell nucleus into the enucleated egg. Briggs and King found that this can be done by sucking an isolated cell into a micropipette that is small enough to break the cell wall but large enough to leave the nucleus still surrounded by cytoplasm. This compromise is required because the nucleus in an unbroken cell does not make the necessary response to egg cytoplasm, and conversely a bare nucleus without surrounding cytoplasm is readily damaged by exposure to any artificial medium. The broken cell with its cytoplasm-protected nucleus is injected into the recipient egg. The amount of donor-cell cytoplasm injected is very small and does not have any effect.
A useful extension of the basic nuc1ear-transplant technique is called serial nuclear transplantation. It involves the same procedure as the one just described except that instead of the donor nuclei being taken from the cells of an embryo or larva reared from a fertilized egg, they are taken from a young embryo that is itself the result of a nuclear transplant experiment. The effect is the same as in the vegetative propagation of plants, namely the production of a clone: a population consisting of many individuals all having an identical set of genes in their nuclei.
One other feature of nuclear-transplant experiments is of the greatest importance for their interpretation. It is the use of a nuclear marker whereby the division products of a transplanted nucleus can be distinguished from those of the host egg nucleus. A nuclear marker is virtually indispensable where attention is to be paid to the development of a very small percentage of eggs that have received transplanted nuclei, since one cannot otherwise be sure that an occasional error in enucleation by hand or by ultraviolet irradiation has not occurred. Only by the presence of a marker in the nuclei of a transplant embryo does one have proof of its origin.
A nuclear marker must be replicated and therefore be genetic. One of the most useful for nuclear transplantation is found in a mutant line of the South African clawed frog Xenopus laevis, discovered at the University of Oxford by Michail Fischberg. The nuclei of most normal frog cells contain two of the bodies called nucleoli; the nuclei of cells carrying the mutation never have more than one. This mutation is almost ideal as a nuclear marker because a sample of cells taken from any tissue at any develop mental stage beyond the blastula (the hollow sphere from which the gastrula arises) can be readily classified as being mutant or not.