Kat Arney

Human Cloning, Part 2 - The Process of Animal Cloning

In the first part of this mini-series we looked at the earliest stages of mammalian development, from the egg and sperm to the ball-like blastocyst. In this second part we turn to the story of cloning, and the technical problems and limitations of the process.

Although cloning burst onto the global media scene with the arrival

of Dolly the sheep in 1997, the technology has been around for decades.

At its most basic the process involves taking an egg cell and replacing

its chromosomes (DNA) with the DNA from another cell, and kick-starting

the 'developmental programme' with chemicals, or an electric pulse

(figure 1). This process is known as Somatic Cell Nuclear

Transfer, or SCNT for short. Hans Spemann first proposed such a

"fantastical experiment" in 1938, and in 1952 tadpoles

were cloned from embryonic frog cells. John Gurdon first successfully

cloned frogs from differentiated (developed) cells in 1962 and since

then the full list of successfully cloned animals has expanded to

include also mice, cows, rabbits, pigs, sheep, cats, horses, zebra

fish and monkeys.

1. Cloning requires an egg cell, and an adult donor cell. The (unwanted) chromosomes are removed from the egg cell and discarded. The nucleus, containing the DNA to be cloned, is removed from the donor cell.		2. The donor nucleus is inserted into the empty egg cell, a process called somatic cell nuclear transfer (SCNT). Afterwards the egg contains a full (adult) set of chromosomes as if it had been fertilised normally.		3. A pulse of electricity, or a chemical 'shock', kick-starts the development process, and the embryo begins to grow.		4. Cell division begins. The subsequent development of the embryo depends upon how successfully the donor nucleus has 're-programmed' the egg.
Figure 1 - The process of embryonic cloning

However, not all clones are created with equal ease. Cloning success

is much more likely if embryonic cells are used as nuclear donors.

These could be embryonic cells taken from an early fetus, or cells

from the inner cell mass of the blastocyst (see Cloning

Part 1). What made Dolly so special was the fact she was the

first mammal to be cloned using a cell taken from an adult - and

even then she was the only live animal to be born out of nearly

three hundred attempts. Since then, other animals have been cloned

from adult cells, but the efficiency of achieving live births is

still extremely low. So what's the big difference between embryonic

and adult cells? The key lies in the concept of epigenetics,

which was introduced in the first part of this series (see also

an earlier article about genetic imprinting).

Cloning requires the reprogramming of an adult or embryonic cell

right back to the very start of development. Adult cells are more

differentiated (specialised) than embryonic cells - they

have made decisions about what sort of cell to be and these choices

are said to be imprinted within the cell. For example, an

embryonic cell has the potential to become all sorts of cell types,

whereas an adult liver cell can only be a liver cell. So embryonic

cells only have to "forget" a few decisions when they

are cloned, whereas an adult cell has been through many more choices

so, essentially, the clock has to be wound back further.

Cells remember what type they are due to epigenetic modifications

of their DNA: molecular tags which mark certain genes as being switched

on or off. These tags are very similar to those on the sperm which

are removed shortly after fertilisation by reprogramming factors

in the egg. In order for an adult or embryonic cell to be reprogrammed

by cloning (by exposing it to the reprogramming environment of the

egg), the epigenetic marks within the DNA must be removed. Adult

cells are believed to have more of these tags than embryonic cells,

so are harder for the egg to reprogramme.

Whether the donor cell is adult or embryonic, a successful clone

must negotiate all the stages of development from one cell to a

blastocyst and then on to being a fully-grown baby. But embryos

can only be grown outside the womb in a test-tube until the blastocyst

stage. After this, the embryo needs to form a placenta and take

resources from the mother. Researchers must transfer the little

balls of cells into the womb of an appropriate surrogate mother,

where it can grow further. In fact, embryos created by mixing eggs

and sperm in IVF clinics are usually transferred before the blastocyst

stage- often when they only consist of two to eight cells.

Experiments in animals such have mice have shown that there is

a high proportion of success in growing cloned embryos up to the

blastocyst stage. Even embryos containing twice the normal number

of chromosomes can grow quite happily to this point, even though

a baby with this many chromosomes could never be born. Yet if these

embryos are transferred into surrogate mothers, very few of them

produce babies. This is because the egg contains many of the ingredients

required for the early stages of development, and will direct the

formation of a blastocyst even if there are severe problems with

the DNA and chromosomes within the embryo. But to grow from a blastocyst

to a baby requires complex patterns of genes being switched on and

off. This can only happen if the DNA of the donor nucleus has been

fully and correctly reprogrammed by the egg during the first stages

of the cloning procedure. So it appears that even if you can create

a clone, and even if that clone makes a blastocyst, you are still

highly unlikely to end up with a baby.

But all is not lost. Blastocysts are extremely useful even if they

don't go on to make a fetus. They are the source of the infamous

embryonic stem cells, or ES cells for short. ES cells are made by

removing the inner cell mass from the blastocyst and growing it

in a dish (figure 2). After a time, a colony of special cells

with almost magical properties appears. These cells have the capacity

to form any tissue of the body, and can be treated with various

chemicals to achieve this. They can also replicate themselves many

times, to create large populations of useful cells. Genetic engineering

techniques work very well in ES cells, allowing mutant genes to

be repaired, and new genes to be added.

1. To make embryonic stem cells (ES Cells), the inner cell mass is removed from a blastocyst.		2. The inner cell mass is transferred to a petri-dish, covered with a nutrient solution, and allowed to grow.		3. The inner cell mass soon develops into a large cell colony which can then be divided into smaller clusters of cells that will themselves continue to grow and multiply.
Figure 2 - How to make Embryonic Stem Cells (ES Cells)

Much weight has been placed on human ES cells as a viable therapy

for many diseases such as Alzheimer's and Parkinson's. Although

we are far from fully understanding the biology of these mysterious

cells, they are certainly brimming with promise. In part three of

this series we'll look at the latest developments from South Korea,

combining ES cell technology and cloning. Are human clones just

around the corner? Or are they already here?

- August 2005

About the Author

Kat Arney is a writer and member of the naked scientists radio programme. She is based at Cancer Research UK