USES AND COMPLICATIONS

Scientists use the word “cloning” to indicate an experimental process by which an exact genetic duplicate is made of a molecule, cell, or organism. It is frequently divided into three general categories. Molecular cloning involves copying genes, short segments of DNA, or cells (sometimes also called cellular cloning) for the purpose of producing multiple copies of a molecule or cell for further scientific study. The cloning of DNA is commonly called recombinant DNA technology or genetic engineering. Cell cloning isolates one particular cell from a large population of cells and places it in an environment where it can grow into a new homogeneous population of cells. Cloning at the organismal level, also called nuclear transplantation, has been used to create genetically identical organisms and has the potential to produce genetically identical tissues and organs from a donor.

The procedure for molecular cloning involves choosing a vector for the study of the target DNA. The choice of the vector depends on the size of the genetic information being studied and whether it is genomic DNA or complementary DNA (cDNA). Common vectors include plasmids (small circular pieces of bacterial DNA), viruses, and artificial chromosomes. For example, if the length of the DNA being studied is small, then the researcher may choose to insert it into a plasmid. By the process of transformation, the selected plasmid is moved into the bacteria (usually E. coli), and as the bacteria divide, cells are produced that are clones for the DNA in the plasmid vector. If the researcher is unsure what area contains the gene to be cloned, then the genome is first fragmented and the individual fragments are inserted into viruses that infect bacteria, or bacteriophages. This creates a library of genetic information. Each bacteriophage vector then infects a bacterium, and the infected bacterial cells will divide to produce a colony of bacteria that contain clones of the genetic information contained within the bacteriophage vector. These colonies may then be screened using molecular techniques to isolate a colony that contains the desired section of DNA.

If the DNA fragment is larger, as is frequently the case with studies of genomic DNA, then the researcher may decide to create an artificial chromosome. The purpose is to create a small, synthetically-derived chromosome that is replicated by the host cell prior to cell division. Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are commonly used, but human artificial chromosomes (HACs) have also been available since 1997. In all cases, the purpose is to create cells that are genetically identical, or cloned, for a specific stretch of DNA. While this is a useful technique, molecular cloning is not able to produce an entire organism that is genetically identical to the original.

In eukaryotic organisms, sexual reproduction produces offspring that contain new combinations of the parents’ genomes. While this provides variability to the species, it complicates medical research since it effectively shuffles the genome every generation. Identical, or monozygotic, twins are the closest thing to clones in humans, but between even their cells small genetic differences exist. For scientific studies of development and cell biology, large numbers of cloned cells are needed.

The process of nuclear transplantation, or cloning, as it is commonly called, has the ability to create large numbers of genetically identical multicellular organisms. In theory, cloning is not a difficult process: Simply remove the DNA from the host cell, replace it with the DNA from the donor cell, and induce the new cell to divide. Procedures such as this have been performed on amphibians since the 1950s, but nuclear transplantation in mammals is slightly more complicated, since mammalian egg cells, or oocytes, are vastly smaller than those of amphibians. Fortunately technological advances in microscopy and embryology have remedied this problem. In mammalian nuclear transplantation, the embryologist uses a microscope equipped with a micropipette. The micropipette is effectively a microscopic needle that is controlled remotely by the researcher. During the procedure, the egg is held in place by a second pipette to allow for greater control. The researcher then inserts the needle from the first pipette into the oocyte through the zona pellucida (outer covering of the oocyte) and gently removes the nucleus from the cell. At this stage, the oocyte contains only the zona pellucida, cytoplasm, and the internal organelles of the cell, such as the mitochondria. The researcher then inserts a donor cell, complete with DNA, into the area between the oocyte and the zona pellucida. At this point, there are effectively two cells alongside each other-one containing nothing but cytoplasm, the other containing the donor DNA. To form a single cell, the plasma membranes of the two cells must be fused. This is done using a process called electrofusion, in which a small current is applied to the cells, temporarily disrupting the membranes and allowing the cytoplasm of the two cells (and the donor DNA) to mix. The end result is an egg cell than contains the DNA of a second cell.

However, this cell is not yet technically a clone. To produce an embryo that is genetically identical to the original donor cell, the oocyte must divide. In some species, the first cell divisions (cleavages) occur readily in response to electrofusion, but this is not always the case. Frequently, growth factors or other chemical signals need to be applied to make the cell divide. All the cells of the blastocyst are clones of one another and the DNA in the original donor cell. Once cell division ensues, the embryo eventually forms a hollow sphere with flat cells on the outside (trophectoderm) and a tight cluster of round cells on the inside (inner cell mass) known as a blastocyst.

Human oocytes begin as cells called “oogonia;” the progenitors of oocytes. During early fetal developing, oogonia divide and increase in numbers, but later in fetal development (18-22 weeks after conception), oogonia cease dividing, undergo one additional round of DNA synthesis and enter the first phase of meiosis, but become arrested in the first phase of meiosis, at which time it is known as a primary oocyte. Like all gametes (sex cells), oocytes undergo a special type of cell division known as meiosis in which the cell divides twice and reduces the number of chromosomes in the progeny cells by half. Primary oocytes remain arrested in the first stage of meiosis from fetal life through childhood until puberty, when a surge of luteinizing hormone from the anterior lobe of the pituitary gland stimulates the resumption of meiosis. This secondary oocyte, as it is now called, arrests at the metaphase stage of the second stage of meiosis and will not complete meiosis unless it is fertilized.

This introduction to the life of human oocytes explains why cloning human embryos has proven to be so technically difficult; the spindle chromosome apparatus that holds the chromosomes in place, poised to complete the second phase of meiosis, is a fragile structure that is easily disrupted by physical or chemical manipulation. Consequently, meiotic arrest in human oocytes is rather unstable and invasive manipulations tend to induce rapid, premature completion of meiosis, which disrupts normal reprogramming of the nucleus. To solve this problem, Shoukhrat Mitalipov and his colleagues at the Oregon Health & Science University treated human oocytes with caffeine to prevent premature exit from meiosis during the cloning procedure, which greatly increased cloning efficiency.

The development of improved technologies in nuclear transplantation has enabled scientists to create organisms that are genetically the same. In the media, the use of the term “clone” usually signifies an organism produced by this procedure. To create a cloned adult organism, the researcher must insert the blastocyst into the uterus of a surrogate mother, who will carry the embryo to term. To date cloned adult sheep, cattle, goats, mules, horses, pigs, mouflons (a wild sheep), mice, rats, dogs, cats, and rabbits have been made in the laboratory. To successfully clone adult animals (reproductive cloning), scientists have adapted the techniques of in vitro fertilization (IVF). IVF retrieves eggs from the mother and fertilizes them with sperm from the father in the laboratory. Subsequently, these embryos are grown in embryo culture, and after they grow to the blastocyst stage, the embryos are transferred into the uterus of the mother. Cloning does not require the fertilization step, since the fusion of the enucleated oocyte with the adult cell takes the place of fertilization. Theoretically, once implanted, the cloned blastocyst should develop in the same manner as an embryo made by means of a natural fertilization event.

A different use of cloning, known as therapeutic cloning, uses cloned embryos (made by means of somatic cell nuclear transfer) to generate a large number of embryonic stem cells for the purpose of treating a disease. Since embryonic stem cells are pluripotent, or have the ability to produce all adult cell types, embryonic stem cells made from a cloned embryo, which are known as “nuclear transfer embryonic stem cells (NT-ESCs) should, theoretically, have the ability to take the place of damaged or diseased cells. Embryonic stem cells (ESCs) are derived from a specific group of cells within the blastocyst called the inner cell mass (ICM). ICM cells are pluripotent cells that can be harvested and grown under laboratory conditions to form an ESC line. Under specific culture conditions, ECSs can differentiate into new tissues, such as nerves, skin, bone, fat, cartilage, or pancreas.

Stem cell scientists have used NT-ESCs to treat various diseases in laboratory animals. For example, researchers from Sloan-Kettering Institute used NT-ESCs derived from cloned mouse embryos to successfully treat animals with a rodent version of Parkinson’s disease. However, the use of blood-making stem cells derived from NT-ESCs to reconstitute the immune systems of mutant mice showed that cells made from NT-ESCs can still be subject to immunological rejection under certain conditions.

Another technique to make stem cells from unfertilized eggs that have been artificially activated has been utilized. Artificial activation of eggs in the absence of fertilization can cause the egg to initiate development, and this process, by which embryonic development begins in the absence of fertilization, is known as parthenogenesis. Parthenogenesis is common in amphibians and insects, but it does not occur naturally in humans. However, if a female egg cell is chemically induced to form a blastocyst without nuclear transplantation, then the resulting stem cells derived from such blastocysts could be used to generate new organs or tissues for the female. Scientists have made several parthenogenic stem cell lines from various animals and humans, and they can be differentiated into several different cell types. However, the stability of parthenogenic stem cells has been questioned by some stem cell scientists. Parthenogenesis cannot be used in males, since sperm cells lack the cytoplasmic components found in egg cells.

INDICATIONS AND PROCEDURES

By definition, the purpose of cloning is to produce genetically identical cells or individuals for scientific studies. Yet even identical (monozygotic) twins, whose cells are derived from the same fertilized egg, are not truly identical. For example, monozygotic twins do not have the same fingerprint pattern, even though they possess the same genes for ridges on the fingers. The reason for this difference is environmental. For twins, the genes establish a general pattern, but it is the touch of the fingers on the inner wall of the uterus that establishes the final pattern of fingerprints. Environment plays a significant role in the development of the embryo, and this fact has presented a challenge for scientists who wish to produce identical genetic clones. Also, the process of DNA replication, while highly efficient, is not perfect, and DNA sequence differences between monozygotic twins and cloned individuals emerge. A study by the Seoul National University compared the genomic DNA sequence of the cloned dog (Snuppy) and the donor animal, and the genomes of two identical Korean twins. Snuppy and his donor (Tai) showed approximately the same degree of DNA sequence differences as human monozygotic twins.

Therefore, Dolly, a cloned ewe, was not an exact copy of her donor, even though she possessed the same genetic information as the donor ewe. According to Ian Wimut, the scientist whose laboratory made Dolly and other cloned sheep, the cloned sheep made in his laboratory were different sizes, and had different temperaments even though they were made from cells that came from the same donor ewe. These differences stem from the fact that Dolly and her “sisters” were raised in the uterus of a surrogate mother and thus were exposed to the minor, but important, environmental variations specific to the surrogate mother. It is known that, by a process called genomic imprinting, the mother can override certain traits in the embryo and impose her own traits, regardless of the genes present in the embryo. The mother also provides all the nutrients needed for the developing embryo, and thus any metabolic problems with the surrogate mother may inhibit proper development in the cloned embryo. Also, particular random events during embryonic and fetal development can generate unique individuals who are also genetically identical.

A potential problem with nuclear transplantation is that not all the DNA in the cell is located within the nucleus. Mitochondria, the energy factories of a cell, contain small circular pieces of DNA. The genes on this DNA are inherited along with the mother’s cytoplasm, so that individuals receive all mitochondrial DNA from the maternal line. During nuclear transplantation, mitochondria are not removed from the host cell, so once transplantation is complete, the new cell contains donor DNA by both host cell and donor cell mitochondria. Because mitochondrial genetic disorders exist, it is known that the genes in the mitochondria contribute to the characteristics of the organism. In fact, in the Korean study mentioned above, the cloned animals had a significantly greater number of mitochondrial mutations.

Cells age and have a finite life span. This appears to be at least partially controlled by the length of the chromosome, specifically the ends of the chromosome called the telomeres. After each cell division, the telomeres shorten like a genetic fuse, until the cell is no longer able to divide. However, work in several species has established that the telomere length of the chromosomes is reset in the cloned embryo.

One of the greatest challenges facing the process of cloning is ethical, which involves the opinion of the general public regarding the cloning of mammals and potentially, humans. Some consider the cloning of organisms to be an unnatural event, while others question the use of cloned embryos as a source of embryonic stem cells. One of the greatest concerns among the general public regarding cloning is the moral right of people to create life by artificial processes. This objection was also applied to IVF when it was introduced in 1978. Another concern is the production of human embryos solely for the purpose of destroying them. The debate over cloning shows no signs of abating and will almost certainly continue into the foreseeable future. The majority of scientists involved in cloning research are interested in either the therapeutic benefits of cell cloning or the study of embryonic development and cell differentiation, and not the creation of a cloned human.

PERSPECTIVE AND PROSPECTS

While for many people the history of cloning may appear to have begun in 1996, when Ian Wilmut of the Roslin Institute in Scotland introduced the world to Dolly the cloned ewe, the reality is that the cloning of organisms had been going on for some time. The making of cloned plants had been occurring for decades and now represents a common occurrence in agriculture. If one restricts the discussion to animals, then Dolly does not really even represent the first cloned mammal, but rather the first adult animal cloned from the cells of another adult animal.

The cloning of animals by nuclear transplantation has its roots in the late nineteenth century, when early embryologists were studying cell division in the eggs of invertebrate animals. The first experiments that transferred a nucleus from one cell to another in a vertebrate animal were conducted in the early 1950s by Robert Briggs and Thomas King. Briggs and King worked with nuclear transplantation in amphibians. These researchers were not interested in the creation of a cloned frog but rather the question of nuclear programming, or whether the cells isolated from the blastocyst had the genetic ability to form a new adult frog. These experiments examined the use of embryonic cells to produce a functionally adult organism. In later experiments, researchers, including Briggs and King, set out to determine at what age of embryonic development cells differentiate to the point where they cannot be used to produce a functioning adult. In essence, they were studying the potency of the cells and beginning to distinguish between totipotent cells and pluripotent cells. For the next several decades, scientists perfected methods of nuclear transplantation in a variety of organisms, including mammals such as mice and rabbits.

In 1996, the researchers at the Roslin Institute used tissue from the mammary gland of an adult ewe in a nuclear transplantation experiment. The result was Dolly, the first mammal to be cloned from an adult cell. Although this experiment was widely reported as producing the first cloned mammal, its real importance was the demonstration that the genes of an adult cell could be expressed in an embryo to produce a living organism. For decades, scientists had debated whether adult cells were capable of being used in cloning. Adult cells are highly specialized, and many of their developmental genes are inactivated. The experiments with Dolly demonstrated that, under the right conditions, the environment within the blastocyst allows DNA from adult cells to be used. In other words, the DNA from differentiated tissue can be used to create undifferentiated stem cells. This remains a major advance in the understanding of cellular processes.

In June 2013, Masahito Tachibana in the laboratory of Shoukhrat Mitalipov at the Oregon Health & Science University reported the derivation of human embryonic stem cells from cloned human embryos. This was the first time human NT-ESCs were produced, and it represented a remarkable technical advance.

In an earlier publication, Mitalipov’s laboratory showed that replacement of an oocyte nucleus by means of somatic cell nuclear transfer, followed by fertilization, can result in a normal embryo. Several scientists suggest that this procedure might allow patients who suffer from mitochondrial genetic diseases to conceive children who do not have these destructive mutations. However, until more animal studies establish the safety of this procedure, it will remain experimental.

The question may then be asked as to why scientists pursue experiments involving the cloning of organisms. Since public opinion is against the cloning of humans and no immediate need exists to clone an individual, then research in this area would appear to be at an impasse. The reality, however, is that the process of nuclear transplantation and organism cloning gives scientists the ability to answer some important questions about cellular differentiation, especially during embryonic development, and the patterns of expression of genes within cells during development.

Furthermore, while the cloning of humans may not be morally acceptable, cloning can serve society in many other ways, such as in studies using transgenic organisms and in tissue and organ transplantation. Scientific research frequently involves the use of transgenic organisms for study. In the study of human genetics and biochemistry, mice are frequently used as a model system. The ability to study the effects of a particular gene in a transgenic organism is dependent on that organism being genetically pure (homozygous) for that trait. In animals, it can take up to fifteen generations of inbreeding to develop a pure line. For organisms with long gestation periods or a small number of offspring per generation, this becomes both cost- and time-prohibitive. The cloning of new organisms that are genetically similar to the donor can facilitate research with transgenic organisms.

Organ transplantation in humans is a difficult process. Recipients of organ transplants must be carefully matched with donors for a variety of biochemical factors to ensure that the new organ is not rejected by the recipient’s immune system. Even when a match is close, the use of immunosuppressant drugs increases the chances of infection in the recipient. The process of nuclear transplantation may alleviate some of these problems. Rather than being matched with a donor, a patient would contribute genetic material for nuclear transplantation. Stem cells could then be harvested and chemically induced to form the required tissue, or someday even the entire organ. Experiments are currently under way to manufacture skin for burn victims using this type of procedure. Even though the discovery of induced pluripotent stem cells have largely marginalized NT-ESCs, many scientists still think that NT-ESCs, which are probably safer than induced pluripotent stem cells, still have an important role to play in research and regenerative medicine. The applications of nuclear transplantation are almost endless, and developments in this area of research have the potential to influence directly the lives of the majority of people alive today.

See also Cytology; DNA and RNA; Embryology; Ethics; Gene therapy; Genetic counseling; Genetic engineering; Genomics; Gynecology; Law and medicine; Multiple births; Obstetrics; Ovaries; Premature birth; Veterinary medicine.