Who has not heard of the famous "Dolly", the Scottish ewe successfully cloned from a somatic cell nucleus? Although frogs and other amphibians had previously been cloned from somatic cells, and cattle from early-stage embryonic cells, Dolly's birth marked a signal achievement, the first live "birth" resulting from a transplanted nucleus that had been extracted from a fully differentiated adult cell.
In order to achieve "Dolly", researchers had to reprogram a fully differentiated (adult) cell back to an embryo-like undifferentiated state. Using highly developed microinjection technology, scientists extracted the donor nucleus from its cell and then transferred it to the receiving cell which had first been stripped of its own nucleus. Dolly was the sole survivor of 277 sheep embryos cloned from a single adult mammary cell. Sometime in the next few months, now that Dolly is old enough to breed, it will be determined whether or not she is fertile and able to bear normal offspring. There is a question, however, as to Dolly's "real" age. Although, as age is commonly counted, she is about two years old, her "genetic" age may be greater. In order to understand why this is, we must understand the function of telomeres.
Somatic (non-germ cell) cell chromosomes are "capped" at their ends by structures called telomeres. Telomeres are repetitive sections of DNA located at the end of chromosomes that make up for a defect in the process that duplicates DNA. Without telomeres to act as caps, essential genes could be lost. In humans, telomeres consist of several kilobases of the telomere repeat 5'-TTAGGG-3'. DNA is duplicated during mitosis by enzymes called polymerases. These enzymes cannot reproduce the very ends of DNA strands, so a cell's chromosomes get shorter each time they get copied. Eventually, the cap is reduced to the point where the DNA can no longer be copied, and the cell dies. Certain cells, such as germ (reproductive) cells and cancer cells can restore their telomere caps, which extends their lives indefinitely. You can think of a telomere as a biological counter which keeps shrinking each time a cell divides through mitosis. When it reaches a critically short length, the chromosome can no longer be copied and the cell dies of old age.
No one knows, at this point in time, if the process of reprogramming Dolly's "ancestor" cell back to an embryonic state reset its telomere "clock." If the clock has not been reset, then Dolly, although about two years old as measured by birthdays, is actually as old as her ancestor (plus 2). What effect this will have on Dolly's ability to breed is unknown.
Other cloned mammals are on the way. Nuclear transfers of somatic cell nuclei have been performed in cattle1 and about 15 genetically identical embryos are currently developing. Two rhesus monkeys have been successfully cloned, using a procedure similar to that used in producing Dolly, but with embryonic cells as nuclei donors. These monkey embryos were cultured briefly in vitro and then transferred to host females. Of 29 embryos so transferred, two live births resulted, a male and a female. Scientists intend to push forward with monkey cloning research, and intend to try other sources of nuclei such as embryonic stem cell or fetal fibroblasts. The goal is to produce large numbers of genetically identical (clonal) monkeys.
There are a number of potential industrial applications of the emerging cloning technology. The production of genetically identical animals will mean fewer should be needed for research purposes and that research can become more accurate when using animals that are genetically homogeneous.
Transgenic research will benefit. Transgenic animals are those that have received a gene from another animal species. For example, goats and mice have been bioengineered so that their genes contain an added human gene that causes them to produce, in their milk, the human protein alpha-1-anti-trypsin. This scarce and expensive protein is vital to patients suffering from congenital emphysema of the lung and cystic fibrosis. Prior to the development of transgenic technology, animal proteins were harvested from donor animal blood. These protein products had the potential to cause severe allergic reactions and required a costly investment in laboratory and purification equipment. The therapeutic protein produced in the milk of transgenic animals is fully human in nature, having been derived from human genes. The fact that these genes now reside in animal genomes is of no consequence. They perform the same task (i.e. encode the identical protein) no matter where they reside, producing fully human alpha-1-anti-trypsin. The process, however, of producing more than one such animal is time consuming2 and fearfully expensive, with fewer than five out of 100 offspring successfully expressing the desired gene. The new cloning technology holds the promise of a low-cost way to produce large numbers of such animals. In fact, once scientists produce the desired transgenic "model" animal, nuclear transfer (cloning) should result in 100% of the offspring of the transgenic "parent" expressing the desired gene; an instant flock of identical transgenic animals.
Once the nuclear transfer technique has matured and scientists can routinely use adult somatic cells rather that fetal/embryonic cells, additional benefits will result. With adult cells as donors, it will be possible to remove undesirable genes, such as the genes (in cattle) that make cow proteins allergenic to some people or to produce infant formula. Another line of research seeks to identify specific proteins that alert the human body that a particular organ is "pig" (as in the case of porcine organ donors) and replace them with "human" ones. To our immune system, if you're not with us, you're against us. The body's ability to recognize its own cells from foreign cells (including cells from most other humans, animals, bacteria, viruses, etc.) is the result of the actions of a group of genes called the MHC (major histocompatibility complex) which holds the plans for several kinds of large proteins. Each person's MHC varies, with only twins sharing identical complexes. The MHCs between different mammal species vary even more, stimulating violent rejection of xenotransplants (organ transplants from one species to another). If a pig can be engineered so that it carries the genes for a human MHC, its organs will be "seen" as human by a human recipient of that organ.
At a recent international symposium conducted in Arlington, VA to review the implications of cloning, it was suggested that cloning technology might be used by infertile couples who might otherwise be unable to have children. Furthermore, cloning (using the nuclear transfer technique) might be a way to reproduce without suffering the risk of disease caused by mutated mitochondrial DNA3. Researchers have known for years that energy metabolism is abnormally low in the brains of patients with Alzheimer's disease. There is a possible genetic explanation in mitochondrial DNA. Most such patients have high levels of a mutant form of cytochrome oxidase, a mitochondrial enzyme that is a key part of the cell's energy-producing machinery. This finding could lead to a diagnostic test for the disease and strongly suggests that this mutation may play a role in actually triggering the neurodegenerative disease.
Ethical issues abound concerning the topic of cloning and public opinion has weighed in against use of the technique on human beings4. Many people harbor the view that the cloning of a human being violates basic tenets of their religions or moral codes. The general consensus in the scientific community is that human cloning should be avoided - at least for now. The process used to create Dolly is not only expensive, but very inefficient (Dolly was the only success in 277 tries). Many find the thought of sacrificing potentially large numbers of human embryos in the quest of a single successful clone ethically abhorrent. When, prior to the Arlington symposium, attendees were polled as to their views on human cloning, most felt that human cloning should be prohibited. At the end of the meeting many had changed their minds and had come to believe that human cloning might be acceptable under certain circumstances. Indeed, a recent N.Y. Times article indicates that people are already becoming desensitized to the issue.
(1) Previous successful cattle cloning experiments, in 1987, involved embryonic donor nuclei.
(2) Until cloning became possible, it was not cost-effective to work with cows because of their long gestation period (12 months), another 12 to reach sexual maturity and another 12 months to lactation, making it a 3-year period until a product could be available for purification.
(3) The cell nucleus is not the only place where DNA can be found. Mitochondria have DNA of their own. There is evidence to suggest that our mitochondria are the descendants of a parasite that once infected the earliest eukaryotic cells and remained in a successful permanent symbiosis.
(4) March 1997 polls by American On-Line and Time-CNN indicated that 56% and 93% of Americans opposed human cloning.