15.7G: Human Cloning
- Page ID
- 5502
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
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.
- 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.