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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         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|   | This staff working paper was discussed at the Council's January 2002 meeting. It was prepared by staffsolely to aid discussion, and does not represent the official views of the Council or of the United States Government.
Staff Working Paper
Scientific Aspects of Human and Animal Cloning
**Introduction**
The following brief review of some scientific aspects of human and animal        cloning is based on scientific research published through the end of December        2001. However, the scientific fields involved in cloning are being very        actively investigated, and significant new developments are being published        frequently. Publication of new results could change some of the interpretations        and emphases in this review. 
Use of unfamiliar technical terms and jargon has been avoided wherever        possible. Scientific names and terms whose use was required are described        and defined in the Glossary of Terms below. In cases where there is disagreement        about definitions, this has been noted. 
**Some Basic Facts about Human Cell Biology and Sexual        Reproduction**
These elementary facts about human cells, germ cells (egg and sperm),        and early embryogenesis will provide the background for understanding        the mechanism of cloning and the differences between sexual and asexual        reproduction. 
Adult human cells with nuclei contain 46 chromosomes, 22 pairs plus two        X chromosomes if the adult is female, or 22 pairs plus one X and one Y        chromosome if the adult is male. These chromosomes contain the bulk of        the cell's DNA, and therefore the genes of the cell. During formation        of sperm cells, specialized cell division (meiosis) produces mature sperm        cells containing 23 chromosomes (22 chromosomes plus either X or Y). During        fertilization of eggs (or oocytes) by sperm, a pronucleus containing half        the egg chromosomes is ejected from the cell. Fusion of egg and sperm        cells and their nuclei (the defining acts of all sexual reproduction)        produces a zygote that again contains a nucleus with the adult cell complement        of 46 chromosomes, half from each parent [See Figure 1]. The zygote then        begins the gradual process of cell division, growth, and differentiation.        After several days in an appropriate nutritive environment, the zygote        attains the 100-200-cell (blastocyst) stage. In normal reproduction, the        blastocyst implants into the uterine wall, where, suitably nourished,        it continues the process of coordinated cell, tissue, and organic differentiation        that eventually produce the organized, articulated, and integrated whole        that is the new-born infant. 
Not quite all the genetic material of a cell resides in its nucleus. Both        egg and sperm cells contain small, energy-producing organelles called        mitochondria. Mitochondria contain a small piece of DNA that contains        the genetic information for making several essential mitochondrial proteins.        When additional mitochondria are produced in the cell, the mitochondrial        DNA is replicated, and a copy is passed along to the new mitochondria        that are formed. During fertilization, sperm mitochondria are selectively        tagged and subsequently degraded inside the cell. Thus, the developing        embryo inherits solely or principally mitochondria (and mitochondrial        DNA) from the egg. 
Human reproduction, in cases hampered by one or another cause of infertility,        has been accomplished with the help of in vitro fertilization of egg by        sperm (IVF) and the subsequent transfer of the early embryo to a woman        for gestation and birth. Though such union of egg and sperm requires laboratory        assistance and takes place outside of the body, IVF reproduction is still        sexual in the biological sense: the new human being arises from two biological        parents through the union of egg and sperm. 
Egg and sperm cells combined in vitro have also been used to start the        processes of animal embryonic development. Implantation of the resulting        blastocysts into the uterus of a female of the appropriate animal species        is widely used in animal husbandry.
**"Reproductive" Cloning (Asexual Reproduction)        of Mammal**
s 
The startling 1997 announcement that "Dolly" the sheep had been        produced by "reproductive" cloning (Wilmut et al, 1997) indicated        that it was possible to produce live mammalian offspring via asexual reproduction        through cloning with adult donor cell nuclei.
1
In outline form, the steps used to produce live offspring in the animal        species that have been cloned to date are (see Figure 1): 
1. Obtain an egg cell from a female of the animal species. 
2. Remove the nuclear DNA from the egg cell to produce an enucleated egg.        
3. Insert the nucleus of a donor adult cell into the enucleated egg to        produce a reconstructed egg. 
4. Activate the reconstructed egg with chemicals or electric current in        vitro to stimulate the reconstructed egg to commence cell division. 
5. Initiate development of the activated, reconstructed egg (zygote) to        a suitable stage of early embryonic development in vitro, then transfer        the embryo to the uterus of a female host that has been suitably prepared        to receive it. 
6. A cloned animal is born that is genetically virtually identical to        the animal that donated the nucleus. However, the mitochondria and mitochondrial        DNA of such a cloned animal would most likely be derived from those of        the egg. 
"Reproductive" cloning carries with it several possibilities        not available through sexual reproduction. Because the number of presumably        identical donor cells is very large, a very large number of genetically        virtually identical individuals could be produced by this process, limited        only by the supply of eggs and female animals that could bear the young.        In principle, any animal, male or female, newborn or adult, could be cloned,        and in any quantity. Because mammalian cells can be frozen and stored        for prolonged periods at low temperature and grown again for use as donor        cells in cloning, one may even clone individuals who have died. In theory,        a clone could be cloned again, on and on, without limit. 
Since the report by Wilmut et al. (1997), attempts have been made to clone        several other mammalian species. As described in more detail below, live        offspring have been produced in a low percentage of cloning attempts with        sheep, cattle, goats, mice and pigs. According to a recent press report        (Kolata, 2001, see 
Appendix A
),        attempts to clone rabbits, rats, cats, dogs and primates using adult cell        DNA have not yet yielded live offspring. While a variety of health problems        have been reported in some cloned animals, surviving cloned cattle appear        physiologically similar to their uncloned counterparts, and two cloned        cows have given birth to their own offspring (Lanza et al., 2001). Several        aspects of the data from the five species that have been cloned are compared        and summarized in Table 1. 
**Human "Reproductive" Cloning**
At this time it is unknown whether any experiments involving human cloning        for reproductive purposes have been carried out. Although statements have        appeared in the press that this was underway (Weiss, 2001), no published        scientific report of any such experiments has been made as of the end        of December 2001. However, the steps in such an experiment would probably        be similar to those described for animal "reproductive" cloning        [see References to Table 1]. A recently reported first attempt at human        "research" cloning has followed the first four steps (Cibelli,        et al, 2001, see Appendix B).
**Human "Research" Cloning**
In vitro production of human embryos through cloning could also be used        by scientists interested in studying the early embryonic forms of human        life, including those produced by cloning. Cloning would make available        embryos that are genetically virtually identical; this would greatly facilitate        comparison of results between different experiments. Specific genes could        be introduced into developing cloned embryos to study the role(s) of these        genes on early human development. Research on cloned embryos is also being        considered as a source of stem cells potentially useful in developing        treatments for degenerative diseases. One such attempt at human "research" cloning has been published        in the scientific literature (Cibelli, et al., 2001) as of the end of        December 2001. It involved the following steps (see Figure 1):1. Obtaining human eggs from informed and consenting female volunteers.        2. Removing the nuclear DNA from the egg cell to produce an enucleated        egg. 3. Inserting the nucleus of a cell from an informed and consenting adult        donor into the enucleated egg produce a reconstructed egg. 4. Activating the reconstructed egg with chemicals to stimulate the reconstructed        egg to commence cell division in vitro. 5. Using a microscope to follow the early cell divisions of the reconstructed        egg. 6. In this experiment, 3 of 19 reconstructed embryos underwent cell division,        but none progressed beyond the six-cell stage. In the "research" cloning experiments described by Cibelli et        al (2001) the stated intent was to create embryos that would progress        to the 100-200 cell (blastocyst) stage, at which point the embryo would        be taken apart, stem cells from the inner cell mass would be isolated,        and an attempt would be made to grow and preserve "individualized"human stem cells (see Figure 2) for the possible future medical benefit        of the donor (NRC/IOM Report). Since the embryos stopped dividing at the        six-cell stage, no stem cells were isolated in these experiments. Although        the steps Cibelli et al (2001) followed up to the blastocyst stage were        the same as those that would be used by those attempting human "reproductive"cloning, Cibelli et al (2001) distinguished their intent from "reproductive"cloning by stating:Strict guidelines for the conduct of this research have been established        by Advanced Cell Technology's independent Ethics Advisory Board (EAB).        In order to prevent any possibility of reproductive cloning, the EAB requires        careful accounting of all eggs and embryos used in the research. No embryo        created by means of NT [nuclear transfer] technology may be maintained        beyond 14 days of development. 
**Parthenogenesis**
It is also possible, using chemical or electrical stimuli, to stimulate        human eggs to undergo several rounds of cell division, as if they had        been fertilized (see Figure 1). In this case, the egg retains all 46 egg        cell chromosomes and egg cell mitochondria. In some animal species this        asexual reproduction process, known as parthenogenesis, has produced live        offspring that contain the same nuclear DNA as the egg. These offspring        are all necessarily female. Cibelli et al (2001) activated eggs obtained from informed and consenting        human donors by parthenogenesis, and obtained multiple cell divisions        up to the early blastocyst stage in 6 out of 22 attempts. Although there        was no report that stem cells were isolated in these experiments, it is        possible that parthenogenesis of human eggs could induce them to develop        to a stage where embryonic stem cells could be isolated. 
**Epigenetic Modification and Reprogramming**
During differentiation of embryonic cells to produce the cells of the        adult, specific chromosomal DNA segments are selectively repressed through        processes called epigenetic modification (see Glossary). In adult cells,        genes whose expression is required only during early embryonic development        are shut down. During the formation of egg and sperm cells, "epigenetic        reprogramming" of genes whose products will be needed during early        embryonic development is required to make these genes active once more.        In cloning using donor nuclei from adult cells, a similar "epigenetic        reprogramming" is required. Based on the data on "reproductive" cloning experiments in animals        in Table 1 and the results of Cibelli et al (2001), most cloned embryos        die either in vitro or after transfer to the uterus. Why is production        of live cloned mammalian offspring a relatively rare event? Several factors        may play a role. Enucleation of the egg may (variably from one attempt        to the next) remove or damage its "epigenetic reprogramming"capabilities. An optimal in vitro nutritive environment for the development        of cloned zygotes may not yet have been determined. One interpretation        (Rideout et al, 2001) attributes the early death of many cloned embryos        to complete failure or incompleteness of "epigenetic reprogramming."At this early stage of experimental work with cloned human embryos, it        is perhaps not surprising that 16 of 19 of the reconstructed eggs did        not undergo cell division and none of the other three reconstructed eggs        divided beyond the six-cell stage (Cibelli et al, 2001). A more detailed        discussion of epigenetic modification and reprogramming as they relate        to cloning will be developed as a component of a broader analysis of "research"cloning. 
**References**Cibelli, J.B. et al., "Somatic Cell Nuclear Transfer in Humans: Pronuclear        and Early Embryonic Development," e-biomed: The Journal of Regenerative        Medicine, 2: 25-31 (2001) [see Appendix        B] Kolata, G. "In Cloning, Failure Far Exceeds Success," New York        Times, December 11, 2001, page D1 [see Appendix        A] Lanza, R.P. et al., "Cloned Cattle Can Be Healthy and Normal,"Science, 294: 1893-4 (2001) NRC/IOM Report - "Stem Cells and the Future of Regenerative Medicine"Rideout III, W.M. et al., "Nuclear Cloning and Epigenetic Reprogramming        of the Genome," Science, 293, 1093-1098 (2001) Weiss, R. "Human cloning bid stirs experts' anger; problems in animal        cases noted," Washington Post, April 11, 2001, page A1 Wilmut, I. et al., "Viable offspring derived from fetal and adult        mammalian cells" Nature, 385: 810-813 (1997) 
**Glossary of Terms***In writing Working Papers for the January 2002 Council meeting,               members of the Council staff have attempted to use terms consistently               as they are defined below in this glossary.***Asexual reproduction**The term "asexual reproduction" means reproduction not initiated        by the union of oocyte and sperm. **Blastocyst**An early stage in the development of mammalian embryos, when the embryo        is a spherical body comprising an inner cell mass that will become the        fetus and an outer ring of cells that will become part of the placenta.        **Biologically related children**Children are "biologically related" to the individuals who are        the sources of their genetic endowment, in the case of sexual reproduction,        to one man and one woman who are the sources of sperm and egg (the "biological"parents), in the case of cloning, to the source of the donor nucleus.        **Cloning***"Reproductive" cloning* -- 1) Obtaining a human or animal        egg cell and removing its DNA. 2) Inserting a nucleus or a cell from a        donor human or animal in order to produce a reconstructed egg that is        genetically very similar to the donor. 3) Implanting the reconstructed        egg in a uterus and delivering the resulting baby or newborn animal. *"Research" cloning* -- 1) Obtaining a human or animal        egg cell and removing its DNA. 2) Inserting a nucleus or a cell from a        donor human or animal to produce a reconstructed egg that is genetically        very similar to the donor. 3a) In some cases, termed "therapeutic"cloning or "cell replacement through nuclear transfer" (CRNT),        the next steps include growing the reconstructed egg to the 100-200 cell        embryo (blastocyst) stage, then taking the embryo apart to isolate and        preserve "individualized" stem cells for immediate or future        use in cell transplantation therapies. 3b) In other cases, the next steps        comprise various scientific experiments designed to better understand        the earliest stages of human embryonic development. *Gene (molecular) cloning* -- Using carrier pieces of DNA (called        vectors) to isolate and characterize DNA segments coding for proteins.        *"Human cloning"* -- The term 'human cloning' means the        asexual reproduction of a new human organism that is genetically virtually        identical to an already existing, or previously existing, human being.        Operationally, it is currently accomplished by introducing the nuclear        material of a human somatic cell into an oocyte whose own nuclear material        has been removed or inactivated, to produce a living organism -- at whatever        stage of development -- that has a human (or predominantly human) genetic        constitution. **Chromosomes**Structures inside the nucleus of a cell, made up of long pieces of DNA        coated with specialized cell proteins, that are duplicated at each cell        division. Chromosomes thus transmit the genes of the organism from one        generation to the next. **Embryo**1. An organism in the early stages of development. 2. In humans, the developing organism from conception until approximately        the end of the second month; developing stages from this time to birth        are commonly designated as fetal. **Epigenetic modification**Turning genes encoded by chromosomal DNA on and off during cell differentiation        through changes in a) DNA methylation, b) the assembly of histone proteins        into nucleosomes, and c) remodeling of chromosome-associated proteins        such as linker histones. **Epigenetic reprogramming**The process of removing epigenetic modifications of chromosomal DNA, so        that genes whose expression was turned off during embryonic development        and cell differentiation become active again. Epigenetic reprogramming        of the donor cell DNA is believed to be an essential process in generating        live offspring through "reproductive" cloning using adult donor        cells and nuclei. **Eugenics**Activity seeking to alter (with the aim of improving) the genetic constitution        of future generations. **Infertility**The inability to conceive a child through sexual intercourse. MitochondriaSmall energy-producing organelles inside of cells. Mitochondria give rise        to other mitochondria by copying their small piece of mitochondrial DNA        and passing one copy of the DNA along to each of the two resulting mitochondria.        **Nuclear transfer**Transferring the nucleus with its chromosomal DNA from one (donor) cell        to another (recipient) cell. In cloning, the recipient is a human egg        cell and the donor cell can be any one of a number of different adult        tissue cells. **Oocyte**As used here, the term oocyte is synonymous with egg. **Parthenogenesis**A form of nonsexual reproduction . . . in which the female reproduces        its kind without fecundation by the male. **Parthenogenote**An embryo that has been produced by parthenogenesis. **Somatic cell (human)**The term "somatic cell" means a diploid cell (having a complete        set of 46 chromosomes) obtained or derived from a living or deceased human        body at any stage of development. **Stem cells**"The definition of 'stem cells' is still a debated issue (Morrison        et al., 1997; Watt and Hogan, 2000). According to the currently prevailing        view (Watt and Hogan, 2000) a stem cell can be defined on the basis of        the following two features: (1) it has an unlimited or prolonged self-renewal        capacity (i.e. the capability to maintain a pool of undifferentiated stem        cells besides giving rise to differentiated daughter cells); (2) it has        uni/multipotency, i.e. the potential to produce one or more differentiated        descendent cell types. . . . Uncertainty that can derive from the terminological ambivalence of the        term 'stem cell' is increased by the misuse of this term, which is often        inappropriately employed (or perhaps voluntarily 'manipulated') in the        popular press to sustain scientific and/or ethical positions for political        ends." (Guena, S., et al, 2001) In this paper, we use the Watt and Hogan (2000) definition given above.        **Zygote**The diploid cell that results from the fusion of a sperm cell and an egg        cell. 
**Glossary References**Guena, S., et al., "Adult Stem Cells and Neurogenesis: Historical        Roots and State of the Art" The Anatomical Record (New Anat.), 265: 132-141 (2001) Morrison, S.J., et al., "Regulatory mechanisms in stem cell biology"Cell, 88: 287-298 (1997) Watt F.M. and Hogan B.L.M. "Out of Eden: Stem cells and their niches"Science, 287: 1441-1446 (2000) 1. Previous experiments dating from the 1950s had shown        that it was possible to clone amphibians. Earlier experiments had also        produced clones of animals using embryonic donor cells. What made the        report of Dolly's birth stand out was the use of adult donor cells and        the fact that a mammal had been cloned. |