How Cloning Developed

Embryo reconstruction by the transfer of a donor nucleus from an adult to an enucleated egg was first proposed in 1938 by a scientist named Spemann. He wondered whether the nuclei of cells change during the development of an early embryo when what start out as uniform-appearing cells differentiate into an enormous variety of different types of cells. The point of Spemann’s suggested study in 1938 was to transfer the nuclei of cells at increasingly advanced stages of an embryo to determine at what stage the potential of the cells to develop into differentiating tissues becomes restricted. This experiment was actually first accomplished in 1952 by Briggs, King, and Martin, who used frogs. Their studies (strictly limited to frogs) were not used to clone an adult frog into another adult frog. That was impossible, and the notion that frogs could be cloned in that way became a popular myth. However, what they did demonstrate is that you could clone a normal fertile adult frog by injecting the nucleus of an early frog embryo (at the blastocyst stage) into a frog egg. That egg would develop into a completely normal adult frog, which was an identical clone of the adult produced by the embryo from which the donor cell was obtained.

In other words, you could not take cells from an adult frog, inject them into an enucleated frog egg, and have, as a result, a healthy cloned adult frog. However, you could take the cell from a very early frog embryo and use the nucleus of that cell to make a clone of that embryo. The nuclei from later-stage embryos (or from an adult) proved to have no developmental potential. Thus, it was believed by legitimate scientists working in the field that although cells of early embryos are totipotential and could be used for cloning, any cells from later-stage embryos, or adults, were considered so differentiated that their nuclei could not possibly be used to clone a new individual.

However, in 1975 it was finally shown, again only in frogs, that a nucleus transferred from an adult skin cell could be injected into an egg and result in what appeared to be normal embryo development. But this embryo completely arrested at the juvenile tadpole stage and never grew into an adult frog. Frogs were popular for these studies because their eggs were so big, and they were much simpler reproductively than mammals, like mice and sheep. Thus, the thinking until the latter 1990s, the era of Dolly, was that frogs were peculiar in the animal kingdom in that they could be successfully cloned from embryo cells and even occasionally from adult cells, but those adult-cell clones would never result in a viable offspring that could grow into a normal adult. But none of these even moderately successful cloning experiments in frogs could be repeated in higher animals. In the mid-1980s it was widely held by leading scientists that the cloning of mammals (such as mice, dogs, cattle, or humans) by simple nuclear transfer was biologically impossible.

McGrath and Softer, of the renowned Wistar Institute in Philadelphia, stated in 1984, “It is now possible to address the question of why nuclei of early mouse embryos are unable to support development while nuclei from much older amphibian [frog] embryos are able to do so.” Early embryo development from fertilization of the egg, up to the eight-cell stage (day three), is independent of the genes of the embryo and completely dependent upon the genes of the egg (from the mother). Genes of the embryo do not take over until the embryo is eight cells. Up until that time, embryo development is being directed by the genes coming from the mother. It was believed in 1984 that frogs and lower animals were different. But that is not true. We now know that the transfer from maternal genome to embryonic genome occurs at the eight-cell stage in humans as well as in most mammalian and lower species, and that frogs were actually easier animals to work with in the early days only because the eggs were so big compared to the eggs of mammals. In fact, the same problems are incurred in cloning frogs as in all other animals, and not a single adult frog has ever been developed from an adult frog cell. Thus, there was no reported cloning of an adult from another adult in any amphibians, reptiles, or mammals prior to the paper on Dolly in 1997.

In that initial experiment, it required 277 nuclear transfers to have a birth of a single normal cloned lamb (from an adult cell) that matured into an apparently normal adult. In contrast, four live lambs resulted from nuclei of embryo cells that were injected into 385 eggs, and two lambs were born from injection of fetal fibroblast cells into enucleated eggs. Thus, very similar to the study in frogs, success rates with cloning were always extremely low, with many abnormal embryos, many miscarriages, many stillbirths, and only a few live, apparently healthy animals. But the success rate was slightly higher (although still dismal) when either fetal or embryo cells were used as the nuclear source, rather than cells from adult animals.

All cells in your body have what’s called a cell cycle. A cell goes from what is called the G-0/G-1 in the early phase (resting), just after cell division, to then synthesizing protein, usually over the course of twelve to twenty-six hours. It is in the early resting phase just after cell division that cloning is possible. Then it once again divides its DNA and replicates itself. After a certain number of divisions, most cells die and cannot divide any further. Stem cells are cells that are continuously replicating, almost eternally, so as to maintain a continued supply of whatever tissue cells are needed. Some types of tissue are rapidly turning over new cells on a daily basis, such as your stomach and intestines, your skin, and your blood cells. These tissues are in a continuous state of wear-and-tear usage and require a great deal of rejuvenation. A good example of this cell turnover is the “healing” that occurs when you have a cut on your hand, or a surgical incision. Fibroblasts and angioblasts move into the open area of a cut, proliferate rapidly, and ultimately form a mature scar that binds together the divided sides of the wound. In a similar manner, the red blood cells that are constantly supplying oxygen to all parts of your body are continuously being broken down and replaced by new red blood cells coming from your bone marrow. The same is true of your white blood cells, which fight infection. Your skin is always wearing down and is being replaced by new cells underneath the skin’s surface. The wear and tear occurring in your intestines, as your food travels through, requires frequent replacement of the lining cells of the intestine in the same fashion.

Other cells of the body replicate much more slowly, or don’t appear to replicate at all. It is these slowly replicating or nonreplicating cells that a team of doctors from Honolulu utilized to go beyond the early Dolly experiments by cloning mice, the most respected of all experimental animals for understanding mammalian biology and human disease. Dr. Yanagimachi’s team in Honolulu had decided that understanding the mystery of healthy and unhealthy embryo development required that cloning be achievable in the mouse model. The mouse, with its rapid reproductive cycle (twenty days), can result in so many generations in such a short time that it is the standard animal for trying to study and understand human disease, much more so than the sheep or any other domestic livestock animal. Yanagimachi decided to try to clone mice not by starving cells into quiescence (the Wilmut approach), but rather by starting with cells that are intrinsically quiescent, i.e., that exhibit little cellular turnover or replication in the adult.