CLONING: How and Why

Written and produced by Bill Stonebarger

"Many of the houses in Roslin, Scotland are built exactly alike. You could call them clones of one another. The unique thing about Roslin, Scotland, however is that right outside of town at the Roslin Institute the very first mammal was cloned. She was named Dolly."

Only a few months later the cloning of mammals took another big step forward when ...

"Here we are Aug. 8, 1997 at ABS Global in DeForest, Wisconsin where they have just announced the cloning of three bull calves. There is one of them - Gene."

In 1998 in Hawaii the first mouse was successfully cloned. In 2000 the first pig was cloned. In 2001 the first cat was cloned. In 2003 the first horse, and in 2005 researchers at the Seoul National University in South Korea cloned the first dog, an Afghan hound. They called her Snuppy, a blend of the SNU (for Seoul National University) and puppy.

What exactly is cloning and why is it important?

Ordinarily living organisms, plant and animal, reproduce themselves by sexual methods. Adult plants or animals produce special sex cells. Females produce egg cells. Males produce sperm cells. Unlike all of the other cells in an adult body, eggs and sperms each contain only half the information needed to produce a new organism. When the egg and the sperm fuse together in sexual fertilization a new fertilized egg results.

This single cell, called a zygote, contains all of the information needed to produce a new and uniquely new organism. Sometimes plants and animals can also reproduce by another method, cloning. In cloning, a non-sexual cell from an adult organism is used to produce a new organism. This non-sexual cell itself contains not half, but all of the information needed. The resulting organism (Dolly for instance) will not be a unique one-of-a-kind organism, but will be almost exactly like the adult whose cell was used for the cloning.

In nature, plants often reproduce themselves by cloning. Farmers and gardeners long ago learned how to use plant cloning methods like budding and grafting to produce new trees, flowers, vegetables and fruit bearing plants. Until now cloning has not been common in animals. Why not?

Embryology, the study of the early stages of development in plants and animals, gives clues.

How does a single cell, a fertilized egg, develop first into an embryo and then how does that embryo develop into a newborn plant or animal?

Like a modern computer, the fertilized egg needs a program, a set of instructions. In a computer the programming is coded in special information language on the computerís chips, hard discs and floppy discs. Much more information is needed to produce a living plant or animal. That information is programmed in living code and it is carried in the nucleus of all living cells.

That information is carried not on chips or discs, but in the form of a chemical structure, deoxyribonucleic acid, DNA for short.

DNA is (for the most part) concentrated in the nucleus of the zygote. It is wound on long spindly structures called chromosomes. A human zygote has 23 pair of chromosomes. Punctuated on each of these chromosomes, like beads on a string, are thousands of genes. It is these genes that carry the actual information to direct the construction process that eventually results in the unique new person.

For the codes to do the job the fertilized egg cell needs the right environment. Specifically, it needs to grow in a salt water environment very similar to that of the ancient ocean. Ordinarily it is the motherís uterus that provides such an environment. Important to our cloning story, however, in very recent years scientists have been able to successfully duplicate the very early stages of this environment in laboratory culture dishes.

When all the environmental conditions are right, the single fertilized egg quivers, doubles its chromosomes, and divides in two. Now you have two cells and each of the two new cells gets the full set of chromosomes, of DNA, of genes. The two cells stick together and the process continues. The two become four, the four become eight, eight become sixteen, thirty-two, sixty-four, etc.

At 5 to 7 days after fertilization, the embryo is still a clump of cells, embryonic stem cells we call them. Each of these hundred or so stem cells has the potential to further divide and then differentiate to form blood cells, muscle cells, bone cells, nerve cells, skin cells. Eventually to become the trillions of cells that make up a human baby. At this very early stage, however, this is only a potential. As yet the embryo has no differentiated cells, no tissues, no organs, no feelings, no consciousness, no experience.

In the past few years scientists have learned how to culture these embryonic stem cells in the laboratory. They have discovered some amazing things about them. For one, they found that given the right chemical environment stem calls can keep living and reproducing indefinitely. They are the only cells we know of, in other words, that are potentially immortal. In the normal natural course of events, embryonic stem cells multiply and very soon the cells that come from them are no longer immortalóand no longer all alike.

The cells in your eye, for example, are not like the cells in your ear. The cells in your lungs are different from the cells in your heart. Where did this difference come from? How did they differentiate themselves from the cells they came from? And why are these heart cells, lung cells, skin cells no longer immortal?

We don't know very much about this process of differentiation yet, but we are learning. And the more we learn about differentiation the more we learn of the secrets to mammal cloning and the secrets to a whole new world of bio engineering. Let's follow the recent research.

As the embryo grows from that single zygote each new cell gets the full complement of genes on its DNA. However, at some point only some of these genes are "turned on." Even though the genes in your skin cells, for instance, have the same genes as your brain cells, your skin cells cannot carry messages, they cannot think. Even though the genes in your bone cells are the same as the genes in your muscle cells, your bone cells cannot contract, your muscle cells cannot deposit calcium or make bone tissue. As cells become differentiated, in other words, they become specialists. They have only a few of their genes turned on. The other genes are there but they are somehow silenced, turned off.

Nature has had a few billion years to learn how to manage these tricks controlling the differentiation of a single fertilized egg into a growing embryo into a new born baby into a mature human being. Until now it has been considered impossible to reverse the process in higher animals.

Biologists said you could never take a mature differentiated cell from a mammal and make it start over again make it behave like an original zygote with all of its genes turned on with no genes silenced.

Never say never. It turned out to be possible after all. In 1996 Ian Wilmut and his colleagues at the Roslin Institute in Scotland discovered how to do it in sheep. Using a method that included controlled cell starvation they were able to reprogram mature cells from an adult sheep's mammary gland and make them behave like one-celled zygotes again. Biologists say they rejuvenated these mature cells, making them totipotent again. This means they now have all of their genes turned on again.

From 277 of these newly totipotent cells they were able to get 30 to develop into embryos. 29 of these embryos were implanted into surrogate mother sheep. 28 of the implanted embryos never made it to birth but one of them did. The researchers named her Dolly. She was the first mammal to be cloned from the cell of an adult mammal. Dolly was born healthy, has developed normally and is now pregnant herself (from using normal sexual methods mating with a suitable ram!).

Other laboratories around the world are now using variations of the Roslin method to clone other mammals. Neal First at the University of Wisconsin-Madison was the first scientist to successfully clone cattle. The average cow, he knew, could produce about fifteen thousand pounds of milk a year. A few super cows, however, could produce as much as 40,000 pounds of milk a year. If he could clone these 40,000 pound supercows it would of great benefit to the dairy industry.

In 1994 he succeeded. In this first cloning he used undifferentiated embryonic cells from supercows which he transplanted into enucleated oocytes of ordinary cows. Oocyte is another word for the female egg. Enucleated means he removed the nucleus from the egg. Now that the researchers at Roslin Institute proved that it was possible to reprogram and use adult cells instead of embryonic cells in this procedure, he and his colleagues are doing just that. With the help of his diagram (and the assistance of his colleagues in his nearby laboratory) he shows and tells just how cloning is done in advanced laboratories like his today.

"What we are doing is superovulating a donor cow. This might be a 40,000 pound cow weíre talking about. And so we would cause her to have not just one egg shed, but ten eggs shed and these eggs would be harvested at about the 30-cell stage. Sort of like this.

"And so we'd flush them actually from the uterus with physiological saline ... flush them into a vessel and retrieve them by looking into the bottom of the vessel with a microscope and finding the eggs.

"Then we would take a single cell from the embryo of 30 cells and we would transfer this into the enucleated oocyte. Now where did the oocyte come from?

"For us the oocycte came from the carcasses of slaughtered cattle. Oocytes are in follicles on the ovaries and we pick those that are in the neighborhood of 2 to 8 mm. in size and not exactly ready to ovulate but they are the eggs that are mature enough to be able to go out and develop.

"And then with a pipette we aspirate the chromatin material in the nucleus from this oocyte we do not push the needle actually into the plasma of the egg. Thatís important. You kill eggs if you do that.

"Then with electro fusion we will fuse this cell that we took over here from the 40,000 pound cow. We will fuse it into the ooplasm of the egg and then we expect the ooplasm to develop them now like it would a brand new embryo back to the 30-cell stage when we could do this all over again.

"The next step for us was to say we can get this to 30 cells. Thatís not very many from a 40,000 pound cow. Maybe it would be more efficient and easier if we took the cells out and put them in a culture dish and multiply them to several thousand cells. We call these embryonic stem cells then. And weíre doing that. And for that we also can make offspring.

"But these are embryonic cells as we said before. What happens for Dolly was that Dolly was the product of a mammary cell. These are adult cells, cells in the cow herself, not an embryo. And these went through a similar process using an oocyte just like we did here, that cell fusion as here, except that it is a differentiated skin cell and now this sort of thing resulted and from this you can go ahead and culture the cells, and more cells like it or you could turn it back and you could serially repeat this process, make more offspring." This is a new field and there are many unanswered questions. For instance: how old was Dolly?

We don't know much about aging in animals. In cloning even though the cell from an adult sheep or cow is reprogrammed to turn on all of its genes, does it retain information about its age? Do the newly born cells in Dolly begin life at five years or a few days? We don't know. Dolly was able to get pregnant at a normal age and to twice deliver normal baby lambs. There was some question, however, whether Dolly would have a shorter life span because she was copied from a 6-year-old animal. And as a matter of fact, Dolly was put down in 2002 when she was 6 years old because of an incurable lung disease which is common in older sheep.

Answers to questions here may give us new insight into the process of human aging.

Another unanswered research problem. Most of the DNA is in the nucleus of a cell, but not all. A small part about 60 genes of the human cell's DNA is carried independently of the nucleus in structures called mitochondria. These mitochondria in cells are responsible for the energy metabolism that cells need to stay alive. In cloning, the nuclear DNA is transplanted from the donor to the newly prepared egg cell, substituting for the eggís original nucleus. But what about the mitochondrial DNA? Very little mitochondrial DNA comes from the donor. So now, the new zygote contains most of its genetic information in the nucleus from an adult donor cell, but a small amount of possibly conflicting? information in the original mitochondria. We donít know yet what this will mean to the newborn cloned animal.

Why are people interested in cloning mammals?

Super cows that produce 50,000 lbs. of milk a year or super sheep that give three times as much wool are a near-term possibility, but probably not the most important long-term advantages to be gained from commercial cloning. For instance, besides Dolly, the scientists at Roslin Institute have recently produced cloned transgenic lambs.

"Transgenic" means lambs that contain some genes from a different species. Polly, one of five lambs cloned from fetal cells in Scotland, carries two human genes as well as her full complement of sheep genes.

Here is how it works. You get cells from a sheep or cow or pig and using new tools like viral transfer, micro-injection or gene guns, you insert genes into the newly cloned zygote or the still undifferentiated embryo (or take genes out!).When the cloned embryo develops into an adult animal it carries this altered gene structure with it.

As an example you could put a human insulin-making gene into a cow embryo. You could engineer it so that this insulin-making gene was only turned on in the mammary glands of the cow, nowhere else. Then when the cow is milked you get not only milk but you get human insulin which you can separate from the milk and use for treating human diabetes.

Similarly you could put genes in cows to produce the blood clotting material people with hemophilia need. Or genes to produce a wide variety and large quantity of many other kinds of pharmaceutical drugs, plasma components, antibodies, hormones and important human nutrients.

You could also use the same method to make animals that have human genetic diseases, like cystic fibrosis or hemophilia or sickle cell anemia. Clone these transgenic animals and then test therapies for the disease before using the therapy on human beings.

It is true that some of this transgenic work has been and is being done today using traditional breeding methods without cloning. Itís not hard to fertilize a cowís egg with bull semen in the laboratory. You can then insert genes into the new zygote or early embryo. You have to wait until the calf is born to tell whether the gene insertion was successful, however. If it was, you let the calf grow to adulthood and breed the resulting cow or bull, Using these methods you gradually develop a herd with the desired genetics. In mammals like cattle or sheep this breeding process takes many years. When and if cloning techniques are reliably available, you will not need to wait those many years. Instead, scientists can grow cells in laboratory culture in large quantities. They can drench these cells with the gene you are interested in inserting. Or you take out genes you donít want. Now you choose only the cells that actually have the desired genetic make-up and use only these cells for the cloning process. When you do get a successfully cloned transgenic cow or bull this way you need not wait for the breeding process to multiply its effectiveness. Instead you can take cells directly from the adult and use them to produce clones. This way you get not one or two but hundreds or even thousands of transgenic cattle or sheep or pigs, all duplicates of the one successfully altered cow or bull.

Already entrepreneurs around the world are confidently investing millions of dollars in these prospects. Another example of cloning and transgenic techniques would be in producing organs for human transplantation. Pigs, for example, have organs that could be used in humans if you could remove the gene or genes that produce proteins that cause the human immune system to reject pig tissues. If you were able to make one transgenic pig with human compatible organs, you could use cloning techniques to produce thousands of these donor pigs.

The most important use in transplantation, though, may not be transplanting entire organs but in transplanting special cells. Dr. First explains how cell cloning may some day soon produce a cure for AIDS and for some cancers.

ďAnother kind of transplantation that perhaps is not so commonly thought about and that is the transplantation of cells. And the best example of this is research on HIV now. One of the more highly promising things on HIV is to engineer blood cells. We call them stem cells. but these are stem cells for the blood lineage are different from stem cells of the embryonic lineage.

ďSo we have embryonic stem cells that will make any and all cells of the embryo. But these are blood cells lineage that will make any and all blood cells. And by starting at that stage and engineering the cells properly one has the ability to create cells that (1) will resist the HIV organism and so it is possible to repopulate the blood in place of the cells that are susceptible and (2) in some cases may be able to destroy the HIV organism.

ďNow what cloning does is provide the opportunity to multiply these cells, engineer the cells in transplantation and with that transplantation that might be blood born tumors or blood cells tumor or it might be a solid state tumor provide it you can get it delivered to the tumor.

ďThe prospects are very exciting and thatís what people are really referring to when the talk about transplantation. And that more than the organ transplant. ď

The biggest question many people ask when they hear of cloning mammals, of course, is what about cloning human beings? Humans are mammals and all of the principles and techniques learned in cow, sheep and other mammal clonings could be used to clone human beings. But will they be used for that? Should they?

The questions are not easy to answer. A kind of human cloning has been going on naturally for thousands of years. About 30 human clones are born in the United States every day! That is because in about one out of every four hundred human births identical twins are born. Identical twins, like clones, have the same DNA, including mitochondrial DNA, in all of their cells. Even so, identical twins are not totally identical. They have the same heredity, but environment and free will also play large roles in making each of us unique.

Experts in human fertility are already helping infertile married couples conceive through various outside-of-the-body fertilization and implantation techniques. Should they be allowed (or encouraged) to use a type of cloning to make pregnancy more probable? They could, for instance, take a human embryo at the stage when it is two or three days old, has only about eight cells, and is still outside of the body in laboratory dish. They could separate these eight cells (each of which still has the potential to differentiate and turn into a human being) and then culture or freeze these cells. If one of the eight did not lead to a successful pregnancy, then you could try another. And another.

Or what about the woman whose ovaries failed to produce eggs. Should she be allowed to use an egg from a donor whose DNA had been removed and replaced with the DNA from a chosen male? Or from herself? In the first case she would give birth to a clone of the male donor. In the second to a clone of herself. What about parents who lose an only child to accident or disease? Should they be allowed to clone this child?

Reproductive cloning is not the only possibility, nor the most likely one to go on in the near future. Another new cloning technique, therapeutic cloning, is much closer to reality today.

New work on embryonic stem cells has opened the door to new breakthroughs in disease and health. Right now scientists are trying to figure out how to trigger embryonic stem cells with the right proteins so that they will turn into heart cells, or liver cells, or nerve cells. Once they succeed a whole new way of treating injury and disease will develop, called therapeutic cloning.

Say a person has heart disease that has left much of his or her heart muscle destroyed. At present the only suitable treatment would be a heart transplant since heart muscle cells never reproduce on their own to repair damage. A major problem with heart transplants is the genetic difference between the donor and the recipient. In order to succeed at all the patient will have to take powerful and dangerous drugs the rest of his or her life to prevent the body from rejecting the genetically foreign cells of that donor heart.

If therapeutic cloning advances, doctors could take a healthy cell from the mouth or skin of the patient, extract the nuclear DNA from one of those cells, insert that DNA in an embryonic stem cell that has had its nuclear DNA removed. In the laboratory dish then they add the correct protein triggers that will cause that stem cell to differentiate and then rapidly reproduce millions of heart muscle cells. They could then inject these cells into the diseased heart where they could continue to grow and replace the diseased and dead cells. And these new heart muscle cells would have exactly the same genetic information as the patientís cells so there would be no chance of rejection.

Perhaps the most important fruit of research in cloning will be basic knowledge about life. Knowing more about how cells differentiate, how genes get turned on and off, is all important. If we learn enough about how cells do this, we may be able, for instance, to devise gene-regulating drugs that would stimulate the growth of nerve cells in the living body. This kind of drug would do wonders for people who are now paralyzed from a spinal cord injury.

We might be able to awaken genes that create bone marrow to help cancer victims fight leukemia. Or stimulate the development of new brain cells in people with Parkinsonís or Alzheimerís disease. One company in Massachusetts has already patented 30 molecules that activate genes responsible for, among other things, the embryonic development of brain, sperm and bone cells.

As you can see, the questions multiply and research is supplying answers at a record pace. Many experts say the 21st century will be the century of biotechnology. As we learn more and more about how to understand and to control life we will need to work just as hard to gain the wisdom needed to make good choices about life.

Within days of the Dolly announcement, the President of the United States set up a special commission to study the legal, ethical and social implications of cloning techniques. This commission made its first report in the summer of 1997.

"At this time it is morally unacceptable for anyone in the public or private sector, to attempt to create a child using somatic cell nuclear transfer cloning," said the report. The commission recommended that Congress enact legislation to legally prohibit such cloning but that the legislation should include a sunset clause. That is, after a period of 3 to 5 years the whole matter could be reviewed and perhaps in the light of new findings new legislation could be drafted. The commission strongly supported continuing research on animal cloning for many of the reasons we have outlined in this program. It also supported continuing research on cloning human DNA and human cell lines.

And finally, one of the most important of the commissionís recommendations was to encourage greater public education and debate about cloning and about all biotechnology. This program is designed to be one small step in that direction.