Oct 21, 2009

Can Human be Cloned?

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.

The cells in an adult are descended from the fertilised egg and 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.

Back in the 20s, the German experimental embryologist Hans Spemann showed that even after 5 divisions of the fertilised 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.

Spemann 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.

Spemann's 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.

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. (However, some other animals have been successfully cloned using nuclei from adult cells.

Why not?
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.

Making Dolly
Enucleate the eggs produced by Scottish Blackface ewes (female sheep). Then treat the ewes with gonadotropin-releasing hormone (GnRH) to cause them to produce oocytes ready to be fertilised. Like all mammals, these are arrested at metaphase of the second meiotic division (meiosis II). After that 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. Then, 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 and to stimulate the resulting cell to begin mitosis (by mimicking the stimulus of fertilisation).
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 fertilisation) and lamb foetuses (26 days after fertilisation). 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. Some scientists have argued that Dolly could have come from a feetal cell that contaminated the mammary gland tissue culture (the cell donor was pregnant at the time). However, two groups have reported (in the 23 July 1998 issue of Nature) that DNA fingerprinting proves that Dolly has a genome identical to the cultured cells and the Finn Dorset ewe that supplied them

Cloning other mammals by somatic-cell nuclear transfer (SCNT)
Since the arrival of Dolly, somatic-cell nuclear transfer (SCNT) has been used to produce seemingly-healthy cows, mice, rats, goats, pigs, rabbits, cats, a mule ("Idaho Gem"), a horse, and a dog.

So what about the prediction (above) that humans will not be cloned from adult cells?
Don't count on it. Great interest is being shown in using somatic-cell nuclear transfer to create embryonic stem cells that could be used to replace missing or defective cells in the body of the nuclear donor.
But the same blastocysts could, in theory, instead be implanted in a uterus to produce a foetus that was a clone of the cell donor.

But keep in mind that humans are not sheep, cows, or mice. In humans, nuclear genes take over earlier in development (at the 4-cell stage) than they do in cattle and sheep (8 cells). Perhaps there will not be enough time for the DNA from an adult human cell to be reprogrammed so that all its genome can once again be expressed.

In monkeys (and presumably in humans), removal of the resident nucleus also removes essential components of the centrosome. Although insertion of the donor nucleus triggers mitosis, the spindles are defective and the resulting cells become aneuploid; that is, fail to receive a correct complement of chromosomes.

Despite these concerns, a group of South Korean scientists have tried to produce cloned human blastocysts by somatic cell nuclear transfer (SCNT) using nuclei from adult human skin cells and placing these in eggs from unrelated women. The blastocysts were to be a source of embryonic stem cells with no attempt made to implant them to create a baby. However, their claims of success appear invalid.