WHAT IT IS

First of all since the matter i will bring up is not easily understandable i'd like to underline some background to let you know the basics of developmental biology. (What follows is the step-to-step process which ended up cloning  the popular and misterious Dolly sheep)

DICTIONARY

oocyte, zygote, differentiated cells:

An oocyte is an unfertilized egg and it has no chance of developing into an animal unless it's fertilized. A recently fertilized egg is called a zygote.  For example, a frog zygote normally divides and grows into a complete animal, a tadpole. Later that tadpole will develop into an adult frog.

A cell from a frog's gut should always remain a frog-gut-cell because it has "differentiated". Differentiation is the natural process whereby cells specialize into a certain kind of cell. As a frog embryo grows and develops its cells differentiate into nerve cells, blood cells, fat cells and many other different kinds of cells. That’s what differentiation is all about.  As a mass of embryo cells divide and differentiate they "create" the animal. The process of differentiation turns zygotes into animals and it's all controlled by the genes. Scientists agree that differentiation must have something to do with changes in the nucleus of cells. The nucleus is the part of the cell containing the genetic material (the DNA all coiled up in organized structures called chromosomes).

The earlier work done by others provided the foundation on which to try something new. Way back in 1975 a fellow named Gurdon developed the method of "nuclear transfer'. This is a two step process-

First he used delicate needles and a good microscope to suck out the nucleus from a frog oocyte, producing an "enucleated oocyte". With the genetic material removed the enucleated oocyte would not divide or differentiate even when fertilized. That was something known. 

But the results from Gurdon’s second step shocked a lot of people. Indeed he used the same equipment and skill to transfer the nucleus from a frog’s gut cell into an enucleated oocyte. That's nuclear transfer, the transfer of a nucleus from one cell to another, creating a "new cell" with a different nucleus. Many of these new cells which Gourdon created behaved like a zygote. They divided and divided  just like a normal developing embryo producing a ball of cells. And this ball of cells differentiated. Nerve cells, skin cells, blood cells appeared just as they would in a normal embryo. After the normal length of time Gurdon had tadpoles. Because the tadpoles had all come from the gut cells of the same adult, they all had the same genetic material. So they were all clones, identical- twins of each other. But unlike normal identical twins they were made from differentiated cells.

Gurdon  proved that differentiation was REVERSIBLE.  Gurdon's method of nuclear transfer made clones from adult cells!  But there were two problems. First, Gurdon's nuclear transferred tadpoles never grew into frogs. Other folks repeated his experiments and got similar results. Nuclear transfer couldn’t clone, frogs from frog cells; all you got were tadpoles, No one knew why. 

The second problem was that Gurdon’s method seemed to work only with frogs ( still "tadpoles'). When scientists tried nuclear transfer with mice, cattle or indeed any mammal, they got nowhere, The 'new cells" sometimes divided a few times, but not for long and none of them differentiated properly. 

The cell cycle is often described as a circle of cell life and division. A cell divides into two "daughter cells" and both of these cells live, "eat", grow, copy their genetic material and divide again producing two more daughter cells. Because each daughter cell has a copy of the some genes in its nucleus, daughter cells are "clones" of each other just like identical twins. This "twining" goes on and on with each cell cycle. This is a natural process.

 Very fast cell cycles occur during development causing a single cell to make many copies of itself as it grows and differentiates into an embryo. Some very fast cell cycles also occur in adult animals. Hair, skin and gut cells have very fast cell cycles to replace cells that naturally die. And cancer is a disease caused by cells cycling out of control. It's no wonder that biologists think the cell cycle is so important.

But there is a kind of "parking spot" in the cell cycle called "quiescense". A quiescent cell has left the cell cycle, it has stopped dividing. Quiescent cells might reenter the cell cycle at some later time, or they might not. It depends on the type of cell. Most nerve cells stay quiescent forever. On the other hand, some quiescent cells may later reenter the cell cycle in order to make more cells. 

Many biologists thought that to make a clone you should transfer the nucleus from a fast dividing cell. It made sense because fast cycling cells are exactly what makes an embryo grow. Besides, the gut cells used to make the tadpole clones were fast cycling cells. Many biologists tried to make clones by transferring the nucleus from fast dividing cells but all of those experiments were unsuccessful.

Dr Campbell  thought about it in a different way. He wondered if a quiescent nucleus would be a better donor. True, it was not cycling (that's what makes it quiescent, by definition) but he thought maybe that's what the nucleus needs for it to be successfully transferred. Maybe the cell needs time to "rest" before starting to make a whole new animal. So they tried it with cells from sheep.

The folks at the Roslin Institute do a lot of work with sheep as part of their partnership with a company called Pharmaceutical Proteins Limited Therapeutics (PPL Therapeutics). Earlier they had made transgenic sheep (sheep with human genes transferred into them).

They used cells from an adult sheep's mammary (breast) glands for the "donor" nucleus. They grew the cells in tissue culture, an artificial situation that is commonly used in laboratories to grow large numbers of cells in bottles. Tissue culture allows scientists to fiddle with the cells and alter their characteristics. That is exactly what Dr Campbell did. He "starved" the cells of important nutrients and the cells stopped growing and dividing. They became quiescent. 

And then ........?

Yes, but creating Dolly was not easy. Using techniques similar to those used 20 years ago by Gurdon, he removed the nucleus from an oocyte that was collected from a Scottish Blackface ewe. (Ewes are female sheep. The Scottish Blackface breed is a common breed of sheep in Scotland easily identified by its black face.)

Oocytes have a "shell" of protein.; and fibers (called the zona pellucida) and it is through this protective coat that Bill injected the nucleus from a quiescent mammary cell into the enucleated oocyte. That cell nucleus was from a different breed of sheep called a Finn Dorset, which happens to be a pure white breed of sheep. He then used a tiny pulse of electricity to cause the new nucleus to fuse with the enucleated oocyte's cytoplasm. (Cytoplasm is the solution inside the cell.) This electricity also helps "kick start cells into 'activity" so they are more likely to divide.

This new, fused cell (containing the Finn Dorset mammary cell nucleus in the cytoplasm of a previously enucleated Blackface oocyte) was transferred into the reproductive chamber of a Blackface ewe (the same breed that provided the oocyte). Biologists repeated this process 276 times! After 148 days, a normal length of time for the Finn Dorset breed of sheep, Dolly was born.as a normal looking Finn Dorset.

Dolly born to a Blackface ewe. This proves that Dolly wasn't the product of a sneaky miting; Dolly's Blackface mom could not produce a white faced sheep no matter who was the father. (It has to do with the genetics of sheep breeds,) But just to be sure, the scientists DNA "fingerprinted" Dolly and her "mom"and proved that Dolly's DNA matched the cells from the tissue culture, not the cells from the ewe that gave birth to her. Dolly is a normal (Finn Dorset) sheep.  You may be surprised to learn that clones had been made at the Roslin Institute before, but those clones were made from the nucleus of embryo cells not adult cells.

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No one knows.  Certainly anyone thinking about trying it would have to find a more efficient rnethod of cloning mammals. You  would also need a lot of women willing to donate both oocytes for the nuclear transfer as well as the use of their uterus in order to grow the clone. Also, there's no guarantee that the same technique would work with humans. Maybe it only works with sheeps.

thanks to Genomics.com