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MAMMALIAN CLONING:
ADVANCES AND LIMITATIONS
Davor Solter
For many years, researchers cloning mammals experienced little success, but recent
advances have led to the successful cloning of several mammalian species. However,
cloning by the transfer of nuclei from adult cells is still a hit-and-miss procedure, and it is not
clear what technical and biological factors underlie this. Our understanding of the molecular
basis of reprogramming remains extremely limited and affects experimental approaches
towards increasing the success rate of cloning. Given the future practical benefits that cloning
can offer, the time has come to address what should be done to resolve this problem.

CLONING EFFICIENCY
Cloning efficiency is calculated
from the percentage of
manipulated embryos that
develops to adulthood, and
reflects how successful or not a
cloning experiment has been.
THERAPEUTIC CLONING
The use of nuclear transfer to
produce individually tailored
human embryonic stem cells
for tissue- and cell-replacement
therapies.
Max-Planck Institute of
Immunobiology, Stbeweg
51, 79108 Freiburg,
Germany. e-mail:
solter@immunbio.mpg.de
The cloning of mammals from adult cells has been
achieved in several mammalian species in the past few
years, indicating that the genome of at least some adult
cells can be reprogrammed to support complete devel-
opment. The mechanisms of reprogramming (as dis-
cussed later in this review) are under intensive investiga-
tion. Now, the discussion about cloning centres on its
commercial use, who owns the cloning patents, and
when the cloning of humans can be expected and con-
demned or condoned. This state of affairs has only
recently been reached; until a few years ago, cloning
from adult cells was but a dream, and twenty years ago,
we were not sure whether cloning by nuclear transfer
would be possible at all.
The commercial use of cloning in agriculture is
imminent. It probably does not require cloning from
adult cells, although this may prove to be useful. Low
CLONING EFFICIENCY (at present less than 1% of nuclear-
transfer embryos develop to adulthood) is not really
an impediment for the agricultural use of cloning
because breeding from a single, cloned, genetically
modified individual should be sufficient. THERAPEUTIC
CLONING may also not be affected by low cloning effi-
ciency because this technique (as discussed below)
does not require a nuclear-transfer embryo to devel-
op to adulthood but only to the blastocyst stage,
which usually has a higher success rate (close to 50%
on average). Low efficiency, however, will affect the
other uses of cloning, and the development of better
cloning methods is one of the next goals of research
in this field.
In this review, I present a brief history of mam-
malian cloning experiments and discuss the present
state of the art and where this may lead. Because numer-
ous books and reviews have been recently written on
this subject16, this review will try to clarify what consti-
tutes fact or fiction with regard to the art of cloning.
Brief history of mammalian cloning
The beginning. The cloning of mammals started with
the development of nuclear-transfer methods in the
mid-to-late 1970s. Derek Bromhall7 transferred morula
nuclei into ovulated rabbit eggs by microinjection or by
fusing blastomeres with ovulated eggs. Fusion was
mediated by inactivated HVJ (haemagglutinating virus
of Japan, also known as Sendai virus), which was at that
time the standard fusion agent for generating cell
hybrids8. This latter approach was inspired by the pio-
neering work of Chris Graham9, who had demonstrat-
ed that somatic cells could be fused to unfertilized and
fertilized mouse eggs and that such fusion products
could develop in vitro to the morula stage (see BOX 1).
Neither Graham nor Bromhall enucleated the recipient
egg, and it is therefore not surprising that these triploid
or tetraploid embryos did not develop very far.
Bromhall reported that a small percentage of injected
or fused eggs lost the egg nucleus by self-enucleation,
and optimistically concluded that [although] no

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development has been demonstrated, there is no
apparent reason why a donor nucleus should not be
able to support development by taking the place of the
egg nucleus in an ovulated egg. Bromhalls work
inspired and provided the scientific basis for one of the
early cloning novels, David Rorviks In his Image: The
Cloning of a Man10. This work was advertised as a true
report of a successful cloning experiment, and
Bromhall sued the author and the publisher,
A a Zygote before enucleation B a Unfertilized egg
Pronuclei Pipette
Zona pellucida
b Enucleation b Enucleation
Pronuclei
in karyoplast
c Nuclear transfer c Nuclear transfer
Nucleus
Or
Karyoplast
Karyoplast
Naked
nucleus
Figure 1 | Current nuclear-transfer procedures. A | A pronuclear-stage, fertilized egg as nuclear
recipient. a | The enucleation pipette is positioned above one of the pronuclei. b | Both pronuclei,
surrounded by a small amount of cytoplasm and plasma membrane, are drawn into the
enucleation pipette. The cytoplasmic bridge is pinched off at the zona pellucida, and the cytoplast
is sealed. c | The karyoplast, or whole nuclear donor cell, is placed under the zona pellucida and is
fused to the cytoplast by electrofusion or inactivated Sendai virus17. B | An ovulated, unfertilized
oocyte as nuclear recipient. a | An oocyte arrested at metaphase of the second meiotic division
with its metaphase plate and spindle at the animal pole. b | The plasma membrane and a small
amount of cytoplasm, which contains the spindle and metaphase chromosomes, are gently drawn
into the enucleation pipette. The cytoplasmic bridge between the cytoplast and karyoplast
eventually breaks and seals the cytoplast. c | The karyoplast, or the entire donor cell, is placed
under the zona pellucida and is fused to the cytoplast by electrofusion or inactivated Sendai virus21.
Alternatively the donor nucleus alone is injected directly into the cytoplast39.
Movie online
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Lippincott, for misrepresenting his results and damag-
ing his scientific credibility. It became apparent during
the trial that the publishers claim of the reality of
cloning could not be sustained, and Lippincott settled
out of court. Interestingly, Bromhall also provided the
visual demonstration of nuclear transfer in another fic-
tional work about cloning, the film The Boys from
Brazil, which was based on Ira Levins book. This film
suggested, for the first time, the need for a clone to
closely imitate the environment and life circumstances
of the original from which it was derived for there to be
a true resemblance between the two.
The next significant experimental step was taken in
the late 1970s and early 1980s, when Jacek Modlinski
showed11,12 that embryonic nuclei could be injected into
a mouse non-enucleated zygote and that a small num-
ber of tetraploid embryos could develop to the blasto-
cyst stage as a result (BOX 1). He also reported12 that when
nuclei from inner cell mass (ICM) cells were injected
into a non-enucleated zygote, tetraploid blastocysts were
observed, but that no embryo developed from the injec-
tion of trophectodermal nuclei.
The first report13 of nuclear transfer into enucleated
eggs was published in 1981. Karl Illmensee and Peter
Hoppe13 claimed that the transfer of ICM nuclei into
an enucleated zygote resulted in live-born mice, where-
as the transfer of trophectodermal nuclei failed to sup-
port development. However, these results, and the tech-
niques that were described, could not be reproduced by
others and have not been to this day1416. Following
these reports, cloning research stalled for a couple of
years. During this time, work in my laboratory focused
on developing a technique to enucleate mouse zygotes
by disturbing the embryo as little as possible this we
did by not penetrating the plasma membrane.
Enucleation was performed by gently sucking out both
pronuclei while retaining them in a small envelope of
cytoplasm and plasma membrane. Donor nuclei were
introduced into the recipient enucleated zygotes by
Sendai-virus-mediated fusion of the KARYOPLAST with the
9
CYTOPLAST, as originally described by Chris Graham .
Unlike Graham, however, we did not remove the zona
pellucida (see BOX 1) but inserted the karyoplast and a
small amount of Sendai virus directly underneath it17
(FIG. 1A). This technique, with certain modifications,
proved to be very reliable, easy to master, and has been
used in nuclear-transfer experiments ever since. This
method was also initially used to show that both the
paternal and maternal genome are required for normal
development and that imprinting (see below) results in
functional differences between parental genomes18,19.
We assumed that this technique would immediately
lead to the cloning of mice, but we were to be disap-
pointed. Although the exchange of pronuclei between
two zygotes resulted in the proper development of
nuclear-transfer embryos, enucleated zygotes that
received a nucleus from a two-cell-stage embryo devel-
oped significantly less well, and those that received
nuclei from four-cell-stage, or older, embryos did not
develop at all20. Fortunately, our negative results and
predictions did not stop others from trying and, two
www.nature.com/reviews/genetics

KARYOPLAST years later, Steen Willadsen published a description of
An isolated donor the first mammalian clones that resulted from the trans-
nucleus, together with its fer of the nuclei of 8- or 16-cell-stage sheep embryos
envelope of cytoplasm
into an enucleated ovulated egg21. He used essentially
and plasma membrane.
the same technique as we did (compare FIG. 1A with
CYTOPLAST FIG. 1B), except that he used as the recipient an oocyte
Enucleated oocyte or embryo that had been arrested at the metaphase stage of the sec-
(zygote) that is used as a ond meiotic division and from which the spindle and
nuclear recipient.
metaphase plate had been removed (FIG. 1B).
POLAR BODY Two predictions could explain our failure and
The structure that is extruded Willadsens success. First, an enucleated oocyte is a bet-
from the oocyte during meiosis, ter recipient than a zygote because it allows more time
which contains one haploid set
for the donor nucleus to adapt and change within the
of chromosomes.
egg cytoplasm before having to support the develop-
BIOREACTORS mental processes; and second, sheep are easier to clone
Animals that are genetically than mice. Extensive work during the next ten years
engineered to produce proteins showed that both predictions, and especially the first,
or macromolecules that are of
use in human medicine.
are true. I will briefly summarize these cloning experi-
ments and refer the reader to several extensive works for
more detail2,5.
Early successes. Soon after the cloning of sheep from
blastomere nuclei, similar results were reported for cat-
tle22, and both of these results have since been repeatedly
confirmed. In addition to generating sheep clones from
nuclei from cleavage-stage embryos, the microinjection
of bovine ICM nuclei into enucleated oocytes also
resulted in live births23. This was, I believe, the first
report of successful cloning using microinjection in
place of karyoplast fusion a point which might be
relevant in current patent disputes (see below). The pos-
sible role of the cell-cycle stage of the donor nucleus in
determining developmental success has also been
explored, and results in rabbits and cows indicate that
nuclei in the G1 phase may provide better results24,25.
Experiments aimed at cloning mice proceeded along-
side cloning studies in larger mammals. The poor devel-
Box 1 | Mouse preimplantation development
The fully grown mouse oocyte arrests at prophase during the first meiotic division
this meiotic division is completed when hormonal stimulation initiates oocyte
maturation and the extrusion of the first polar body structure. The haploid egg arrests
again at metaphase during the second meiotic division. At this stage, the 20 mouse
chromosomes are aligned on the metaphase plate, which is localized at one pole of the
egg, perpendicular to the plasma membrane. The spindle body, which is composed of
microtubules that attach to the chromosomes at their centromeres, lies parallel to the
plasma membrane. Following fertilization, the egg completes the second meiotic
division and extrudes the second polar body. In the fertilized egg (zygote), egg and
sperm chromosomes undergo DNA synthesis and form the male and female pronuclei.
On completion of DNA synthesis, the pronuclear membranes dissolve, the pronuclei
fuse and the first cleavage division is completed. Subsequent cleavage divisions result in
embryos that comprise 4, 8, and 16 blastomeres a 16-cell-stage embryo is known as a
morula owing to its characteristic form. When fluid starts to accumulate between the
blastomeres, resulting in a fluid-filled cavity called the blastocoel, the embryo becomes
a blastocyst. The blastocyst has an outside cell layer, called the trophectoderm, inside of
which, and to one side of the blastocoel, is a small group of cells called the inner cell
mass (ICM). The blastocyst hatches from a proteinaceous envelope, known as the zona
pellucida, and implants into the uterus. Once implanted, the trophectoderm gives rise
to extraembryonic membranes, whereas the ICM gives rise to the entire embryo and
also contributes to the extraembryonic membranes. Growth of the ICM gives rise to
the egg cylinder in rodents and to the embryonic disc in humans and sheep.
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opment of embryos when enucleated zygotes were used
as recipients was repeatedly confirmed26,27, and the use of
enucleated two-cell-stage cytoplasts as recipients proved
to be much more successful26,28,29. Enucleated oocytes
were also used successfully, and the role of the cell-cycle
stage of the donor cell in improving cloning efficiency
was established3033. In all these experiments, only the
nuclei from cleavage-stage embryos were used as donors.
The successful use of donor nuclei derived from ICM and
trophectoderm cells required the development of serial
nuclear transfer14, a technique in which donor nuclei are
initially fused with enucleated oocytes and allowed to
develop to the two-cell stage. These two-cell-stage
embryos are then enucleated and the removed nuclei are
fused with enucleated normal two-cell-stage cytoplasts.
In the most successful application of serial nuclear trans-
fer, four-cell-stage mouse nuclei arrested at metaphase
were fused with enucleated oocytes and allowed to devel-
op into two diploid nuclei by preventing the extrusion of
the POLAR BODY34. These diploid nuclei were then trans-
ferred into enucleated zygotes17, which were cultured to
the blastocyst stage and then transferred to foster moth-
ers. This procedure is very efficient and resulted in the
birth of one sextuplet (with 75% cloning efficiency) and
several quadruplets (FIG. 2).
Cloning from adult cells. As the use of blastomere-stage
nuclei to generate clones slowly improved, it became
apparent that this would not be very useful for commer-
cial agricultural purposes. Two goals of commercial
cloning the production of genetically superior ani-
mals with desired phenotypic properties and the pro-
duction of genetically modified animals to serve as
BIOREACTORS would only be achieved if cloning could
be done from adult cells (to allow an adult phenotype to
be selected) or from established cell cultures (to enable
genetic engineering). The first indication that some-
thing like this might be possible came from the success-
ful cloning of calves by using nuclei derived from cul-
tured ICM cells35. Provided that the length of time in
culture from establishment until nuclear transfer was
less than a month, using these cells as nuclear donors
resulted in the birth of live calves.
At that time, the idea that the success of cloning
depended on the donor cell being somehow embryonic
in nature still predominated. However, this viewpoint
changed radically when sheep were successfully cloned
from nuclei taken from a cultured cell line36. This cell line
was derived from an embryonic disc (see BOX 1), but it
was an established cell line that had been through several
passages36 and the cells did not resemble those of any
previously described embryonic stem (ES)-cell lines.
This success suggested that our ideas as to what consti-
tutes a good nuclear donor for cloning might have been
significantly off the mark, and that cloning from differ-
entiated, even adult, cells might be imminent37. Wilmut
and colleagues proved this prediction to be correct38, and
the birth of lamb 6LL3 (Dolly see link to the Roslin
Institute) marked the beginning of an era in which
cloning ceased to be an interesting subject just for mam-
malian developmental biologists and suddenly became
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Nuclear donor Nuclear recipients
Enucleation Enucleated zygotes
Transfer of donor nucleus
Activation
DNA synthesis
Karyokinesis Diploid
nuclei
Figure 2 | Serial nuclear transfer34. The donor nucleus is fused with the enucleated
ovulated oocyte (as in FIG. 1B). This can be considered to be the reprogramming phase
of the procedure. The egg is then activated and nuclear division takes place while CYTOKINESIS
is inhibited. It is likely that both nuclei undergo DNA synthesis and are presumably
reprogrammed. They are then transferred into two enucleated zygotes (as in FIG. 1A) to
capitalize on the better developmental potential of a correctly activated zygotic cytoplasm.
the concern of politicians, ethicists, theologians and the
general public. Following the birth of Dolly, the cloning
of mice39 (FIG. 3) and cows40 from adult cells dispelled any
doubts that cloning from adult cells was possible41.
Current methodological considerations
Choice of nuclear donor. The nuclear-transfer methods
used at present are illustrated in FIG. 1. As mentioned
before, the most important condition for the success of
nuclear transfer is enucleation without penetration of
the plasma membrane. This can easily be accomplished,
regardless of whether a zygote (FIG. 1A) or an oocyte (FIG.
1B) is used as the nuclear recipient. The optimal choice
CYTOKINESIS
The division of the cytoplasm
of a nuclear donor is not quite so clear. Many different
of a parent cell into daughter cells isolated from adult tissues and from established cell
cells after nuclear division. lines have been used and, whereas some have proven to
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be unclonable, it is not clear why cells from some tissues
make better nuclear donors than those from others. It is
quite possible that the unclonability of certain adult cell
types is more apparent than real. For example,
Wakayama and colleagues39 failed to obtain live mice
following the transfer of nuclei from Sertoli cells or
brain cells. Recently, however, the successful cloning of
mice from immature Sertoli cells has been reported42,
and it might be that the cloning of nuclei from adult
neurons will follow. The clonability of adult cells is of
considerable biological interest (see below), but it has
little significance for the practical application of cloning,
for which it is quite sufficient that only certain adult
cells be clonable.
Status of donor nucleus. Data from Wilmut and col-
leagues36,38 indicate that the donor nuclei should come
from quiescent cells arrested in the G0 phase of the cell
cycle, but this requirement, although it may be benefi-
cial, is not absolute. It is probably important to know
the exact state of the donor nucleus in terms of DNA
content. The recipient cytoplasm of the enucleated
oocyte imposes on a donor nucleus a behaviour that
mimics that of the removed genetic material. Soon after
transfer of a diploid but 2n nucleus, the chromosomes
condense (FIG. 4a) and eventually become organized on a
metaphase plate (FIG. 4b). As these chromosomes are
composed of a single chromatid and a single centro-
some, they cannot divide and are randomly pulled
towards one or the other spindle pole. At this point, it is
crucial that cytokinesis or extrusion of any genetic
material from the egg is inhibited. The chromosomes
decondense and form several pseudo-pronuclei (FIG. 4c),
and DNA synthesis begins. Once DNA synthesis is com-
pleted, the chromosomes are ready for division the
first mitotic division takes place and development pro-
ceeds. The behaviour of a diploid but 4n nucleus is simi-
lar, except that once the metaphase plate is formed, the
chromosomes can divide and the egg can complete the
second meiotic division and extrude the pseudo-second
polar body43. In this case, extrusion of the genetic
material is essential to prevent tetraploidy. Once the
nuclear content is again 2n, DNA synthesis can begin
and development can proceed, as in the previously
described case. It is clear that successful cloning requires
that the manipulator knows the status of the donor
nucleus to determine whether to inhibit or to allow the
extrusion of the pseudo-second polar body. It also
seems that it is much better (or possibly essential) for
the donor nucleus to be in either the G0/G1 or the G2
phase of the cell cycle but not to be in-between. It may
also be essential that one round of DNA synthesis takes
place in the oocyte before the first cleavage division.
Status of oocyte. The quality of the recipient oocyte
might also affect the cloning outcome, but there is very
little one can do about oocyte selection at present. It is
likely that forced maturation by various superovulation
protocols results in a certain number of oocytes that
cannot sustain development. Unfortunately, in most
cases, there is no way of avoiding superovulation
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Figure 3 | The first mouse cloned from an adult somatic

(cumulus) cell. The mouse, called Cumulina, at four weeks b
of age, next to her white-coat-coloured foster mother.
(Figure reproduced with permission from REF. 39 (1998) Macmillan
Magazines Ltd.)

because it is required to obtain a sufficient number of

eggs (or eggs at the desired time), and we have no objec-
tive criteria for selecting good from bad oocytes,
except by morphology. Following nuclear transfer, the
oocytes have to be activated to proceed with develop-
ment, and we know that ACTIVATION and fertilization are
not functionally equal. For example, downregulation of
the inositol-1,4,5-trisphosphate receptor that normally
follows fertilization does not occur following oocyte
activation by any of the activation protocols used at pre-
sent44,45. The origin, frequency and amplitude of CALCIUM
TRANSIENTS that follow sperm entry into normally fertil-
c
ized eggs are definitely altered in in vitro activated
oocytes46. However, in vitro activated eggs must develop
after nuclear transfer or cloning would indeed be
impossible, but one can easily surmise that the absence
of normal fertilization could contribute to the low levels
of nuclear transfer success.

Ideal cloning practice. If we are to make an educated

OOCYTE ACTIVATION
This occurs when the binding
of sperm to the egg cell
membrane triggers a series of
responses in the oocyte that
prepare the oocyte for
fertilization and block the entry
of more sperm.
CALCIUM TRANSIENTS
A series of repetitive oscillations
in calcium concentration that
move across the egg cytoplasm
following sperm entry, which
are essential for egg activation.
guess as to the best cloning method, several points can
be made with reasonable confidence. As mentioned
above, gentle enucleation without penetrating the plas-
ma membrane is vital. Various cell types can be tested
as nuclear donors and the best one selected. For most
purposes, stable diploid cell lines that can be genetically
modified in culture are preferable, and these can
include ES cells or fetal fibroblasts. The method for
introducing the donor nucleus is not critical and vari-
ous fusion methods (such as using inactivated Sendai
virus or electrofusion), as well as direct microinjection,
have been successfully used. (If oocyte activation is
required to occur simultaneously with fusion, then one
should use electrofusion methods rather than inacti-
vated Sendai virus.) As discussed above, it is probably
essential to use an oocyte as the recipient if nuclear
reprogramming is to take place. However, a zygote
makes a much better recipient in terms of it being
properly fertilized and not artificially activated. One
possible way around this dilemma is to use the serial
nuclear-transfer approach34, and researchers will have
to balance the extra work involved in serial transfer
Figure 4 | The behaviour of a nuclear donor following its
transfer into an enucleated oocyte. a | Soon after injection,
the chromosomes condense and, b | following egg activation,
are organized into a structure that resembles the metaphase
plate. c | Once segregated, the chromosomes are organized
into two or more pronuclear-like structures called pseudo-
pronuclei. Owing to the block of cytokinesis, all pseudo-
pronuclei remain in the oocyte and undergo DNA synthesis.
Once the cytokinesis block is removed, the egg completes
its first cleavage division and initiates development.
(Figure reproduced with permission from REF. 39 (1998) Macmillan
Magazines Ltd.)
against the anticipated benefits. Alternatively, better
ways of oocyte activation might become available in the
future, and a recent report that shows that the microin-
jection of nitric oxide recapitulates the events of nor-
mal egg activation47 is a promising start.

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In addition, each species presents its own unique set
of technical problems for cloning research. The recent
success in cloning pigs probably stems from the transfer
of a very large number of manipulated embryos (25)
into a single recipient48, because several fetuses are nec-
essary to secure a pregnancy in the pig. As only one live
clone was recovered (out of 110 embryos transferred
into several recipients), it is possible that some other
unknown factor(s) contributed to this success. In
another report of successful pig cloning, the investiga-
tors used both serial nuclear transfer, as described by
Kwon and colleagues34, and the transfer of a large num-
ber of nuclear-transfer embryos (22100) into a single
recipient49. They obtained a single litter of five piglet
clones from 72 transferred embryos. Six other recipients
receiving a similar number of embryos failed to become
pregnant or to maintain a pregnancy. It is again not
clear what exactly contributed to this single success.
These results confirm that we are far from understand-
ing the technical complexities of mammalian cloning.
Why is cloning so inefficient?
Differential activity of maternal and paternal
genomes, and the results presented here, suggest that
the cloning of mammals by simple nuclear transfer is
biologically impossible.
Jim McGrath and Davor Solter20
Everybody loves to quote the above sentence, usually in
its abbreviated (italicized) and slightly misleading form
to demonstrate how wrong scientific predictions can be.
How wrong were we even to suggest the impossibility
of cloning? Cloning is obviously possible but it is also
inefficient (TABLE 1), and the associated problems are not
all of a technical nature. Once nuclear transfer is com-
pleted, the chances of the cloned embryo developing into
a healthy adult are about 1 in 100. Many elements could
contribute to this rate of failure, and it is unclear which
of them could be eliminated by technical improvements.
It is likely that the methods used to activate eggs follow-
ing nuclear transfer are not optimal (as discussed) and
that several events that follow fertilization by sperm do
not occur properly in activated eggs, for example, the
rate and amplitude of calcium transients. Co-injecting
sperm extracts or purified molecules once they are
known together with a donor nucleus might alleviate
this problem47. Alternatively, serial transfer using an enu-
cleated zygote as the ultimate recipient34 may be benefi-
cial. We also know relatively little about the roles that fail-
ure of imprinting and nuclear reprogramming play in
causing cloning experiments to fail, and these aspects
need to be investigated in the future.
Imprinting. By using nuclear transfer to construct
embryos that contained only maternal (gynogenones)
and only paternal (androgenones) pronuclei, we18 and
others19 established that maternal and paternal genomes
are not functionally identical and that both are neces-
sary for normal development. It is now clear that certain
genes are imprinted during gametogenesis so that only
the paternal or only the maternal allele is expressed after
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fertilization50,51 and that normal development requires
the correct expression of imprinted genes. The imprint-
ing mark is not really understood in molecular terms,
and this makes it difficult to analyse the imprinting sta-
tus of imprinted genes in adult cells. Nevertheless,
imprinting must be preserved in some of the adult
nuclei used as donors, at least to a sufficient degree to
allow development. The same is true for reprogram-
ming, except that we know even less about how repro-
gramming is accomplished and how successful it is. One
could design experiments in which both imprinting and
reprogramming are tested; the ability to clone mice
from established cell lines makes these experiments pos-
sible, although very difficult. Ideally, we should examine
the imprinting status of the donor nucleus in a cloned
cell line and track imprinting changes following nuclear
transfer. The problem with such an experiment, howev-
er, is that we do not know for certain what the imprint-
ing mark is, and we can only judge the imprinting status
by the expression of the imprinted genes, which may be
very misleading in preimplantation embryos in which
most imprinted genes are expressed biallelically. One
could, of course, look at the methylation status of genes
that have differently methylated maternal and paternal
alleles that reflect their imprinted status. However, the
difficulty with this approach is that one would need to
look not only at successfully developing late fetuses and
adults, but also at the imprinting status of several
imprinted genes in a single early nuclear-transfer
embryo. In this way, the frequency of nuclear-transfer
embryos that have normal imprinting at these genes
could be determined, although such an analysis is
almost impossible to do on an individual preimplanta-
tion nuclear-transfer embryo at this time.
Reprogramming. For cloning to work at all, the donor
nucleus must be reprogrammed in the eggs cytoplasm
following its transfer, which means that it must cease its
own programme of gene expression and assume an
expression programme typical of a zygotic genome.
There is also a time limit to reprogramming it must
be completed by the time that the normal activation of
the embryonic genome would have taken place (activa-
tion of the embryonic genome occurs one to a few days
after fertilization, depending on the mammalian
species). In normal development, both the oocyte and
sperm nuclei are transcriptionally silent at the time of
fertilization; their chromatin then undergoes extensive
remodelling, accompanied by the activation of the basic
transcription machinery52,53, to result in the activation
of the embryonic genome. Following nuclear transfer,
the situation is somewhat different. Donor nuclei are
not transcriptionally silent before transfer, and the mol-
ecular composition of their chromatin is likely to be dif-
ferent from that of egg and sperm nuclei. Kikyo et al.54
recently demonstrated the redistribution of nuclear
proteins following the exposure of nuclei from a
Xenopus cell line to a Xenopus egg extract. This work
represents a first step in a long-term effort to under-
stand the biochemistry of nuclear remodelling and
genome reprogramming.
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Table 1 | Cloning efficiency HETEROPLASMY might also contribute to the failure of

nuclear-transfer embryos. Experimental work to
Species Nuclear donor Number of oocytes Live births/adults
cell type manipulated (%)* address the basic biology of cloning is at its very begin-
Sheep Embryonic epithelium36 244 5/2 (0.8)
ning and, although this knowledge is not absolutely
Mammary epithelium38 277 1/1 (0.4) essential for the practical application of cloning, it will
Fetal fibroblasts TR70 507 6/4 (0.8) certainly affect its widespread use.
Fetal fibroblasts TR68 417 14/3 (0.7)
Cow Cumulus cells40 99 5/2 (2.0) Cloning humans
Oviductal cells40 150 3/2 (1.3) The cloning of humans for reproductive purposes is
Fetal fibroblasts TR69 276 4/3 (1.1)
Adult fibroblasts71 1,103 6/4 (0.4) either legally forbidden by some countries (see links to
Senescent fibroblasts58 1,896 6/6 (0.3) European ban on human cloning and the United States
Mouse Cumulus cells74 1,345 16/10 (0.7) National Bioethics Advisory Commissions recommen-
Adult fibroblasts75 717 3/1 (0.4) dations) or where there are no specific laws against it
ES cells43 1,765 5/1 (0.05) is under a voluntary moratorium. Nevertheless,
ES cells F1 (REF. 43) 1,087 26/13 (1.2)
ES cells76 418 8/0 offers of substantial sums of money have been made by
ES cells F1 (REF. 76) 227 7/7 (3) people who wish to pay for the privilege of being
Pig Fetal fibroblasts48 110 1/1 (0.9)|| cloned, and many such offers crowd the Internet. There
Adult granulosa cells49 401 5/5 (1.2)|| is no evidence to suggest that the cloning of humans
*Represents clones surviving to adulthood as a percentage of total number of manipulated from adult cells is impossible, and I would not be sur-
oocytes. Nuclei were derived from an R1 cell line. Number of embryos actually transferred into prised to hear of it being attempted in the future. One
surrogate sows. Number of manipulated oocytes was 23 times higher. ||Calculated on the basis
of embryos transferred. (ES, embryonic stem; TR, transgenic.) would hope that the current technical difficulties
involved in securing a large number of egg donors and
surrogate foster mothers would be a sufficient deterrent.
An ideal analysis of reprogramming would involve Most cloned embryos fail to develop, and about half
evaluating the expression of, maybe, 100 genes, 50% of of those that are born die soon after birth (TABLE 1). The
which are turned on in the nuclear donor but are off in negative consequences of cloning could be significantly
the normal preimplantation embryo, the other half delayed until after birth, and although no discernible
being the opposite. Following nuclear transfer, we should negative behavioural effects have been observed in adult
examine each embryo individually and determine how cloned mice, a significant increase in their body weight
many of the genes that should have been turned off are has been reported63. The current number of adult mam-
actually turned off, and how many of the genes that mals that have been cloned is probably too small to
should have been turned on are turned on. We have detect all the possible negative effects of the technology,
started to identify the genes that are turned on in early and it is quite possible that several, unpredicted, nega-
preimplantation embryos55 and to develop the methods tive consequences of cloning will be observed in the
to analyse reliably the expression of numerous genes in a future. On the basis of these results, the safety issue has
single cleavage-stage embryo, so we should be able to been the paramount concern behind the decisions to
determine the degree of reprogramming with some con- ban human reproductive cloning. That is fine for now,
fidence. It is clear from the limited amount of available but what will happen if the safety issues can be resolved
data that, in most nuclear-transfer embryos, reprogram- in the future? The reason the safety issue was used in the
ming is incomplete, resulting in death during develop- first place was because various advisory board members
ment and even after birth56,57 (TABLE 1). could not agree as to why the cloning of humans should
be forbidden. If one is to view reproductive cloning as
Telomeres. Because telomeres shorten as somatic cells part of an ever-expanding human reproductive free-
divide, and because it is assumed that this shortening dom, an outright ban of it cannot be justified. It is likely
contributes to cellular ageing, the use of adult cells as that every society and, ultimately, every person, will
nuclear donors could result in cloned animals that have decide independently. Although there is a basis for the
prematurely shortened telomeres and presumably a current heated debate and emotional response, I do not
reduced lifespan. At present, it is unclear whether the believe that the issue will be of any major significance in
transfer of an adult nucleus causes cloned animals to have the future. Reproduction by cloning can hardly ever
shorter telomeres, and contradicting results have been replace the current tried and true methods.
reported58,59. Wakayama et al.60 cloned mice for six gener- Although reproductive cloning is off limits at pre-
ations and reported no evidence for significant telomere sent, there is increased pressure to allow so-called thera-
shortening. Whether the observed inability to clone past peutic cloning. Ever since human ES cells have been iso-
the sixth generation is because of the accumulation of lated64,65, therapeutic cloning has been seen as their
genetic errors, or whether the experimental design optimal use66. In this procedure, the nuclei from a
favours cells with long telomeres, remains unclear. patients cell would be transferred to an enucleated
oocyte, and the resulting blastocyst would be used as a
MITOCHONDRIAL
Mitochondria. In normal development, all mitochon- source of autologous ES cells, as has been recently
HETEROPLASMY
The presence of more than one
dria are maternally derived, whereas some61, but not described using cloned mouse blastocysts67. These
type of mitochondrial DNA all62, the cloned animals contain mitochondria from would be used in tissue grafts to avoid immunological
within the same cell. both the nuclear donor and the recipient. MITOCHONDRIAL reactions against the grafted tissue. There is a growing

NATURE REVIEWS | GENETICS VOLUME 1 | DECEMBER 2000 | 2 0 5

2000 Macmillan Magazines Ltd
REVIEWS

belief that differentiated derivatives of ES cells will even-
tually be used to treat many genetic and degenerative
diseases. If these expectations prove to be correct, and
autologous cells derived by therapeutic cloning seem to
be the best approach to use, I expect that many will
overcome their moral objections to producing and then
destroying cloned blastocysts.
Conclusions
The main practical purpose of cloning is to generate
genetically modified farm animals to serve as bioreac-
tors. Therapeutically valuable proteins produced by
these animals can be secreted in milk or deposited in tis-
sues and organs from which they can be extracted1.
Attempts to produce such animals by classical transgen-
esis (that is, by injecting DNA into pronuclei) has met
with little success, as have the numerous attempts to iso-
late ES cells from large farm animals for transgenesis.
So, the current and only way to produce genetically
modified, large, farm animals is to genetically engineer
cells from cultured cell lines and to introduce these
modifications by nuclear transfer68.
Substantial investments have been made, untold rich-
es are anticipated, and the first glimpse of success is visi-
ble1,4,6871. No wonder, then, that the companies involved
are fiercely contesting the ownership of patents to pro-
tect the different cloning methods72. I cannot see many
novel aspects in the different cloning techniques that fol-
lowed on from earlier reports17,21. Wilmut and
colleagues38 consider the quiescent state of the donor cell
to be crucial, but this may not be the case, as already dis-
cussed. Microinjection methods using a piezo microin-
jector were pioneered by Wakayama and colleagues39,
but other fusion methods are probably equally efficient73
and less difficult to master. Maybe only the method of
serial transfer, as recently described14,34 (FIG. 2), represents
a novel technical development, and this method could be
crucial for targets that have, until now, proved difficult to
clone, such as primates, rabbits and pigs. However, this
method is technically demanding and will only be used
in extreme cases. It will be interesting to see how legal
contests regarding the significance of these minor tech-
nical differences will be resolved.
The cloning of mammals is a fascinating biological
problem, although it is difficult and attempts at it are
rarely successful. Learning more about its basic mecha-
nisms will teach us much about the control of gene
expression and the genetic control of development. The
practical use of cloning in agriculture has already
become a reality, and the relatively low success rate
should not be a problem for this particular application
of cloning. The reproductive cloning of humans is likely
to cause more individual concern than real societal
effect, as it is unlikely to become a widespread method
of reproduction even if possible and safe. The future
application of human therapeutic cloning and of ES
cells in tissue and cell therapy will be determined by its
usefulness it would be shortsighted to reject it out of
hand until we learn more about its possible future role
in human medicine.
Links
DATABASE LINKS Inositol-1,4,5-trisphosphate receptor
FURTHER INFORMATION In his Image: The Cloning of a
Man | Roslin institute | European ban on human cloning
| United States National Bioethics Advisory
Commissions recommendations |
ENCYCLOPEDIA OF LIFE SCIENCES Nuclear transfer
from established cell lines | Imprinting in mammals |
Cleavage and gastrulation in mouse embryos | Sperm-
egg binding in mammals

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Acknowledgements
I would like to thank Professor Ryuzo Yanagimachi from the
University of Hawaii for kindly giving permission to reproduce
Figures 3 and 4.
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