This staff working paper was discussed at the Council's January 2002 meeting. It was prepared by staff
solely to aid discussion, and does not represent the official views of the Council or of the United States Government.
Scientific Aspects of Human and Animal Cloning
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
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
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)
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
"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
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
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.
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"
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
Kolata, G. "In Cloning, Failure Far Exceeds Success," New York
Times, December 11, 2001, page D1 [see Appendix
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.
The term "asexual reproduction" means reproduction not initiated
by the union of oocyte and sperm.
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.
"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
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.
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.
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.
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.
Activity seeking to alter (with the aim of improving) the genetic constitution
of future generations.
The inability to conceive a child through sexual intercourse.
Small 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.
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
As used here, the term oocyte is synonymous with egg.
A form of nonsexual reproduction . . . in which the female reproduces
its kind without fecundation by the male.
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.
"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.
The diploid cell that results from the fusion of a sperm cell and an egg
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.