In February 1997, a research team at the Roslin Institute in Edinburgh, Scotland, headed by Dr. I. Wilmut, reported (in the 27 February 1997 issue of Nature) that they had succeeded in producing a healthy lamb, named Dolly, from the nucleus of a cell taken from an adult sheep.
Why has this achievement created such a stir?
After all, all the cells in an adult are
- descended from the fertilized egg
- have been produced by mitosis
So each cell in the adult would be expected to carry the complete diploid complement of genes of the organism.
Figure 188.8.131.52 Gray crescent
Many years earlier, the German embryologist Hans Spemann showed that even after 5 divisions of the fertilized egg, the nuclei retained the potential to program the complete development of an adult. Using strands of baby hair, he tied loops around fertilized amphibian (newt) eggs so that they were constricted into two halves with the nucleus confined to one half and a narrow bridge of cytoplasm connecting the two halves.
He found that:
- At first only the half containing the zygote nucleus would divide by mitosis.
- Eventually a nucleus would cross into the other half and it, too, would begin dividing.
- So long as both halves contained some of a cytoplasmic region called the gray crescent, the second half would then go on to develop into a second perfectly-formed embryo.
So even at the 32-cell stage of development, the nuclei had not lost the potential to program the complete development of the organism. His results suggest, then, that cloning should be possible. Genetically-identical nuclei should be able to produce genetically-identical individuals. And, of course, that is what occurs in human identical twins, triplets, etc. They are "miniclones".
Since Spemann's time, the development of micromanipulators has made it possible to remove nuclei from cells. To test the developmental potential of these nuclei, they can then be injected into "enucleated" eggs; that is, eggs whose own nucleus has been removed or destroyed. Using this technique (called somatic-cell nuclear transfer or SCNT), it was found that the nucleus of any of the thousands of cells of the frog blastula is able to guide perfectly normal development when transplanted into a frog egg lacking its own nucleus.
Figure 184.108.40.206 Clones of tadpole
Furthermore, when a transplanted nucleus has programmed the formation of a new blastula, the cells of the new blastula can then serve as the source of identical nuclei to use to form a clone of genetically-identical tadpoles.
Up to now, adult cells have not worked. When nuclei from adult frog cells are transplanted into enucleated eggs, the results have not been so successful. Many of the resulting embryos are abnormal and stop developing. Despite years of effort, no adult frogs have been produced by nuclei transplanted from adult frog cells. And until Dr. Wilmut's success, no other animals had been successfully cloned using nuclei from adult cells.
No one knows for certain. But what is known is that during development, the DNA of differentiated cells does change — not in its sequence — but in its ability to be expressed (transcribed). The DNA becomes chemically altered. As many as 8% of the cytosines (C) in an organism's DNA become methylated. Genes containing methylated DNA are inactive. So it appears that although every cell in the adult organism contains the entire genome, many of the genes can no longer be expressed.
What Dr. Wilmut's group has done is find a way to unlock the full potential of gene expression in the nuclei of cells taken from an adult mammal. They do not know the biochemical basis of their achievement, but this is how they did it.
Figure 220.127.116.11 How Dolly was made
- Enucleate the eggs produced by Scottish Blackface ewes (female sheep).
- Treat the ewes with gonadotropin-releasing hormone (GnRH) to cause them to produce oocytes ready to be fertilized. Like all mammals, these are arrested at metaphase of the second meiotic division (meiosis II).
- Plunge a micropipette into the egg over the polar body and suck out not only the polar body but the haploid pronucleus within the egg.
- Fuse each enucleated egg with a diploid cell growing in culture.
- Cells from the mammary gland of an adult Finn Dorset ewe (they have white faces) are grown in tissue culture.
- Five days before use, the nutrient level in the culture is reduced so that the cells stop dividing and enter G0 of the cell cycle.
- Donor cells and enucleated recipient cells are placed together in culture.
- The cultures are exposed to pulses of electricity to
- cause their respective plasma membranes to fuse;
- stimulate the resulting cell to begin mitosis (by mimicking the stimulus of fertilization).
- Culture the cells until they have grown into a morula (solid mass of cells) or even into a blastocyst (6 days).
- Transfer several of these into the uterus of each (of 13, in this case) Scottish Blackface ewes (previously treated with GnRH to prepare them for implantation.
- Wait (with your fingers crossed).
The result: One ewe gave birth (148 days later) to Dolly.
What made Dolly different?
The Wilmut group also used the same technique to produce healthy lambs using cells from lamb embryos (9 days after fertilization) and lamb fetuses (26 days after fertilization). But in these experiments, there was no way to know the phenotype of the nuclear donor because it had not yet been born. So, too, the recent cloning of monkeys from embryo nuclei represents simply an expansion of nature's ability to produce identical twins, etc. whose traits we will not know until they are born and grow up. But the nucleus that made Dolly came from an adult animal whose phenotypic traits were there to be seen.
How do we know that Dolly is not the progeny of an unsuspected mating of the foster mother?
- She has a white face and the foster mother is a Scottish Blackface
- DNA fingerprinting reveals bands found in Finn Dorset sheep (the breed that supplied the mammary cells), not those of Scottish Blackface sheep
What accounts for this remarkable achievement?
Besides years of hard work, we do not know. Perhaps:
- Using cells in G0 demethylates inactive genes and makes it possible once again for them to be expressed.
- The mammary gland cells were not truly differentiated epithelial cells but primitive stem cells present in the mammary gland.