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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.
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 development. The mechanisms of reprogramming (as discussed later in this review) are under intensive investigation. 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 condemned 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 nucleartransfer 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 efficiency because this technique (as discussed below) does not require a nuclear-transfer embryo to develop 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 mammalian cloning experiments and discuss the present state of the art and where this may lead. Because numerous books and reviews have been recently written on this subject16, this review will try to clarify what constitutes fact or fiction with regard to the art of cloning.
Brief history of mammalian cloning

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 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 pioneering work of Chris Graham9, who had demonstrated 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

Lippincott, for misrepresenting his results and damaging 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 fictional 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 number of tetraploid embryos could develop to the blastocyst 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 injection 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, whereas the transfer of trophectodermal nuclei failed to support development. However, these results, and the techniques 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 disappointed. 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 developed 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

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years later, Steen Willadsen published a description of the first mammalian clones that resulted from the transfer of the nuclei of 8- or 16-cell-stage sheep embryos into an enucleated ovulated egg21. He used essentially the same technique as we did (compare FIG. 1A with FIG. 1B), except that he used as the recipient an oocyte that had been arrested at the metaphase stage of the second meiotic division and from which the spindle and metaphase plate had been removed (FIG. 1B). Two predictions could explain our failure and Willadsens success. First, an enucleated oocyte is a better recipient than a zygote because it allows more time for the donor nucleus to adapt and change within the egg cytoplasm before having to support the developmental processes; and second, sheep are easier to clone than mice. Extensive work during the next ten years showed that both predictions, and especially the first, are true. I will briefly summarize these cloning experiments 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 cattle22, 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 possible 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 alongside cloning studies in larger mammals. The poor develBox 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.

KARYOPLAST

An isolated donor nucleus, together with its envelope of cytoplasm and plasma membrane.
CYTOPLAST

Enucleated oocyte or embryo (zygote) that is used as a nuclear recipient.

POLAR BODY

The structure that is extruded from the oocyte during meiosis, which contains one haploid set of chromosomes.
BIOREACTORS

Animals that are genetically engineered to produce proteins or macromolecules that are of use in human medicine.

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 transfer, four-cell-stage mouse nuclei arrested at metaphase were fused with enucleated oocytes and allowed to develop into two diploid nuclei by preventing the extrusion of the POLAR BODY34. These diploid nuclei were then transferred into enucleated zygotes17, which were cultured to the blastocyst stage and then transferred to foster mothers. 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 commercial agricultural purposes. Two goals of commercial cloning the production of genetically superior animals with desired phenotypic properties and the production 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 something like this might be possible came from the successful cloning of calves by using nuclei derived from cultured 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 constitutes a good nuclear donor for cloning might have been significantly off the mark, and that cloning from differentiated, 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 mammalian developmental biologists and suddenly became

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Nuclear donor Nuclear recipients

Enucleation

Enucleated zygotes

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 colleagues36,38 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 beneficial, 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 centrosome, 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 completed, the chromosomes are ready for division the first mitotic division takes place and development proceeds. The behaviour of a diploid but 4n nucleus is similar, 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

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

CYTOKINESIS

The division of the cytoplasm of a parent cell into daughter cells after nuclear division.

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 of a nuclear donor is not quite so clear. Many different cells isolated from adult tissues and from established cell lines have been used and, whereas some have proven to

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a

Figure 3 | The first mouse cloned from an adult somatic (cumulus) cell. The mouse, called Cumulina, at four weeks 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 objective criteria for selecting good from bad oocytes, except by morphology. Following nuclear transfer, the oocytes have to be activated to proceed with development, 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 present44,45. The origin, frequency and amplitude of CALCIUM TRANSIENTS that follow sperm entry into normally fertilized 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 guess as to the best cloning method, several points can be made with reasonable confidence. As mentioned above, gentle enucleation without penetrating the plasma 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 various 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 inactivated 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

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

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 pseudopronuclei. Owing to the block of cytokinesis, all pseudopronuclei 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.)

A series of repetitive oscillations in calcium concentration that move across the egg cytoplasm following sperm entry, which are essential for egg activation.

against the anticipated benefits. Alternatively, better ways of oocyte activation might become available in the future, and a recent report that shows that the microinjection of nitric oxide recapitulates the events of normal 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 necessary 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 investigators used both serial nuclear transfer, as described by Kwon and colleagues34, and the transfer of a large number 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 understanding 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 completed, 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 following 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 enucleated zygote as the ultimate recipient34 may be beneficial. We also know relatively little about the roles that failure 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 necessary 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

fertilization50,51 and that normal development requires the correct expression of imprinted genes. The imprinting mark is not really understood in molecular terms, and this makes it difficult to analyse the imprinting status 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 reprogramming, except that we know even less about how reprogramming 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 possible, 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, however, is that we do not know for certain what the imprinting 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 preimplantation 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 (activation 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 molecular composition of their chromatin is likely to be different 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 understand the biochemistry of nuclear remodelling and genome reprogramming.

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might also contribute to the failure of nuclear-transfer embryos. Experimental work to address the basic biology of cloning is at its very beginning and, although this knowledge is not absolutely essential for the practical application of cloning, it will certainly affect its widespread use.

Table 1 | Cloning efficiency

Species Sheep Nuclear donor cell type Embryonic epithelium36 Mammary epithelium38 Fetal fibroblasts TR70 Fetal fibroblasts TR68 Cumulus cells40 Oviductal cells40 Fetal fibroblasts TR69 Adult fibroblasts71 Senescent fibroblasts58 Cumulus cells74 Adult fibroblasts75 ES cells43 ES cells F1 (REF. 43) ES cells76 ES cells F1 (REF. 76) Fetal fibroblasts48 Adult granulosa cells49 Number of oocytes Live births/adults manipulated (%)* 244 277 507 417 99 150 276 1,103 1,896 1,345 717 1,765 1,087 418 227 110 401 5/2 (0.8) 1/1 (0.4) 6/4 (0.8) 14/3 (0.7) 5/2 (2.0) 3/2 (1.3) 4/3 (1.1) 6/4 (0.4) 6/6 (0.3) 16/10 (0.7) 3/1 (0.4) 5/1 (0.05) 26/13 (1.2) 8/0 7/7 (3) 1/1 (0.9)|| 5/5 (1.2)||

HETEROPLASMY

Cow

Cloning humans

Mouse

Pig

*Represents clones surviving to adulthood as a percentage of total number of manipulated oocytes. Nuclei were derived from an R1 cell line. Number of embryos actually transferred into surrogate sows. Number of manipulated oocytes was 23 times higher. ||Calculated on the basis of embryos transferred. (ES, embryonic stem; TR, transgenic.)

An ideal analysis of reprogramming would involve evaluating the expression of, maybe, 100 genes, 50% of which are turned on in the nuclear donor but are off in the normal preimplantation embryo, the other half being the opposite. Following nuclear transfer, we should examine each embryo individually and determine how many of the genes that should have been turned off are actually turned off, and how many of the genes that should have been turned on are turned on. We have started to identify the genes that are turned on in early preimplantation embryos55 and to develop the methods to analyse reliably the expression of numerous genes in a single cleavage-stage embryo, so we should be able to determine the degree of reprogramming with some confidence. It is clear from the limited amount of available data that, in most nuclear-transfer embryos, reprogramming is incomplete, resulting in death during development and even after birth56,57 (TABLE 1). Telomeres. Because telomeres shorten as somatic cells divide, and because it is assumed that this shortening contributes to cellular ageing, the use of adult cells as nuclear donors could result in cloned animals that have prematurely shortened telomeres and presumably a reduced lifespan. At present, it is unclear whether the transfer of an adult nucleus causes cloned animals to have shorter telomeres, and contradicting results have been reported58,59. Wakayama et al.60 cloned mice for six generations and reported no evidence for significant telomere shortening. Whether the observed inability to clone past the sixth generation is because of the accumulation of genetic errors, or whether the experimental design favours cells with long telomeres, remains unclear.
MITOCHONDRIAL HETEROPLASMY

The presence of more than one type of mitochondrial DNA within the same cell.

Mitochondria. In normal development, all mitochondria are maternally derived, whereas some61, but not all62, the cloned animals contain mitochondria from both the nuclear donor and the recipient. MITOCHONDRIAL

The cloning of humans for reproductive purposes is either legally forbidden by some countries (see links to European ban on human cloning and the United States National Bioethics Advisory Commissions recommendations) or where there are no specific laws against it is under a voluntary moratorium. Nevertheless, offers of substantial sums of money have been made by people who wish to pay for the privilege of being cloned, and many such offers crowd the Internet. There is no evidence to suggest that the cloning of humans from adult cells is impossible, and I would not be surprised to hear of it being attempted in the future. One 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. Most cloned embryos fail to develop, and about half of those that are born die soon after birth (TABLE 1). The negative consequences of cloning could be significantly delayed until after birth, and although no discernible negative behavioural effects have been observed in adult cloned mice, a significant increase in their body weight has been reported63. The current number of adult mammals that have been cloned is probably too small to detect all the possible negative effects of the technology, and it is quite possible that several, unpredicted, negative consequences of cloning will be observed in the future. On the basis of these results, the safety issue has been the paramount concern behind the decisions to ban human reproductive cloning. That is fine for now, but what will happen if the safety issues can be resolved in the future? The reason the safety issue was used in the first place was because various advisory board members could not agree as to why the cloning of humans should be forbidden. If one is to view reproductive cloning as part of an ever-expanding human reproductive freedom, an outright ban of it cannot be justified. It is likely that every society and, ultimately, every person, will decide independently. Although there is a basis for the current heated debate and emotional response, I do not believe that the issue will be of any major significance in the future. Reproduction by cloning can hardly ever replace the current tried and true methods. Although reproductive cloning is off limits at present, there is increased pressure to allow so-called therapeutic cloning. Ever since human ES cells have been isolated64,65, therapeutic cloning has been seen as their optimal use66. In this procedure, the nuclei from a patients cell would be transferred to an enucleated oocyte, and the resulting blastocyst would be used as a source of autologous ES cells, as has been recently described using cloned mouse blastocysts67. These would be used in tissue grafts to avoid immunological reactions against the grafted tissue. There is a growing

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belief that differentiated derivatives of ES cells will eventually 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 bioreactors. Therapeutically valuable proteins produced by these animals can be secreted in milk or deposited in tissues and organs from which they can be extracted1. Attempts to produce such animals by classical transgenesis (that is, by injecting DNA into pronuclei) has met with little success, as have the numerous attempts to isolate 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 riches are anticipated, and the first glimpse of success is visible1,4,6871. No wonder, then, that the companies involved are fiercely contesting the ownership of patents to protect the different cloning methods72. I cannot see many novel aspects in the different cloning techniques that followed 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 discussed. Microinjection methods using a piezo microinjector 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 technical 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 mechanisms 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 | Spermegg binding in mammals

1. 2. 3. 4. 5.

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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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