1999/11/01 by Robert G. McKinnell, Marie A. Di Berardino · 2 citations
Biochemistry, Genetics and Molecular Biology · Medicine · #Animal Genetics and Reproduction #Pluripotent Stem Cells Research #Reproductive Biology and Fertility
paper · pdf · doi:10.2307/1313647
openalex publication_date 1999/11/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/06/23
Although the method of nuclear transplantation should be valuable principally for the study of nuclear differentiation, it may also have other uses. (Briggs and King 1952, p. 462) Clearly, 1997 was the year of the clone (Figure 1). The cover illustration of Nature (27 February 1997) announced the birth of Dolly, the ewe cloned from an adult sheep in Scotland, and Science (19 December 1997) proclaimed Dolly to be the “breakthrough” of the year. Newspapers, news magazines, radio, and television were even more fervent in their reports of Dolly. Even now, several years later, Dolly (Wilmut et al. 1997) and a number of mice (Wakayama et al. 1998, Wakayama and Yanagimachi 1999) and calves (Kato et al. 1998, Wells et al. 1999), all cloned from adult cells, continue to evoke fascination. Dolly, the sheep produced from the transfer of a nucleus of an adult mammary gland cell at the Roslin Institute in Scotland. Was Dolly the first animal to be cloned? Of course not. Why, then, the sudden, almost unprecedented attention to the announcement of a cloned sheep? We believe that because Dolly was the first animal cloned from an adult cell, she stimulated scientists, theologians, ethicists, journalists, and politicians to contemplate the application of cloning to humans. The point of this article is not to reconsider the extension of cloning to humans, a subject that has already been covered (e.g., Silver 1997, Kolata 1998, Nussbaum and Sunstein 1998), but to consider the genuine rationales that stimulated the original and continuing efforts in cloning research. Cloning was never intended as a procedure for the simple multiplication of animals. Frogs are cheap in the United States, as are sheep in Scotland. The reasons for cloning are more complex than simply producing identical animals, and in this article we consider those reasons and the results that have been obtained with the procedure. We place cloning in the historical context of developmental biology and review results obtained with the procedure. Some readers may wonder how two scientists with cloning experience view the ethics of human cloning. We offer our views in the epilogue. How do cells become specialized during development? One cell, the zygote, gives rise to a multiplicity of cells that in time become increasingly specialized, or differentiated. In the latter part of the nineteenth century, August Weismann believed that differentiation results from the differential and sequential partitioning of the genome as the cells divide (reviewed by Wilson 1928, Spemann 1938). The attractiveness of the now-discarded Weismann hypothesis was that it could be tested. Wilhelm Roux, in an 1888 experiment (Spemann 1938), killed one cell (blastomere) of a two-cell amphibian embryo and found that a half-embryo developed, suggesting that some genes are lost during cell replication. However, in 1892, Hans Driesch found that if the blastomeres of two-cell sea urchin embryos were physically separated, entire embryos formed from each blastomere (Spemann 1938); similar results were obtained when amphibian blastomeres were isolated, provided that they contained a portion of cytoplasm known as gray crescent material. Thus, the genome was not diminished, but rather reproduced during cell division. (Roux's half-embryos were later interpreted to result from the inhibitory effect of the dead blastomere.) CLONING HAS PROVIDED INSIGHTS INTO NUCLEAR DIFFERENTIATION, NUCLEAR REPROGRAMMING, CELLULAR AGING, AND GENOMIC IMPRINTING Blastomere separation of embryos beyond the 2- to 16-cell stage (depending on species) was noninformative for testing genomic potential because the cells had too little cytoplasm. But in 1894, Jacques Loeb fortuitously observed that fertilized sea urchin eggs sometimes ruptured when exposed to hypotonic solutions (Spemann 1938). The extruded portion of the egg was usually bereft of a nucleus and remained uncleaved. Occasionally, nuclei traversed the isthmus between the cleaving egg and the extruded cytoplasmic material. In such cases, the extruded cytoplasm cleaved along with the main mass, resulting in the formation of two entire embryos. In 1914, Hans Spemann performed a conceptually identical experiment on an amphibian egg (Spemann 1938). He constricted a zygote with a noose made of baby hair, causing the egg to assume the shape of a dumbbell. When the cleaving (nucleated) portion reached the 8- or 16-cell stage, he loosened the constriction and permitted a nucleus to move to the non-nucleated cytoplasmic portion. Here, too, the non-nucleated portion cleaved and formed a clone of its nuclear donor. These primitive nuclear transplantation (cloning) experiments affirmed the view that the complete genome is replicated during cell division, at least during early cleavage. The stage was now set for modern cloning experiments to examine the genomic potential of older embryonic cells. However, Spemann (1938) “could see no way for the moment” to manually insert a nucleus from older embryos into enucleated cytoplasm. Robert Briggs and Thomas J. King found a way. Around 1943, Briggs (1911-1983), an embryologist working at what is now the Fox Chase Cancer Center in Philadelphia, wanted to determine whether the genome of older somatic nuclei remains equivalent to the zygote nucleus throughout development (Patterson n.d.). One possible approach was to transplant a somatic nucleus into an oocyte whose own nucleus had been removed and then observe what type of development occurred. Briggs was aware that Comandon and de Fonbrune (1939) had transplanted single nuclei in the unicellular amoeba, but he recognized that nuclear transfer in the oocytes of metazoans would be more arduous because of their complex physiological and biochemical requirements for embryonic development. Over the next 7 years, while analyzing the development of haploid and triploid frog embryos, he became experienced with various microsurgical techniques that would contribute to the procedure of nuclear transfer. In 1948, one of us (Di Berardino) joined Briggs's laboratory and was there to witness the first cloning of metazoan animals in 1952. In 1949, Briggs began searching for a research fellow to develop a nuclear transplantation procedure for the North American leopard frog, Rana pipiens. He also applied to the National Institutes of Health for funds to support the project. His first attempt was rejected because the reviewers considered his proposed research a harebrained scheme with little chance of success. However, a second application was successful, and Briggs brought King in as his research fellow in 1950. The two scientists first determined that eggs lacking a functional nucleus but containing a cell division center developed at best into partial blastulae (Briggs et al. 1951). This baseline study established the total developmental potential of the recipient host; any further development could, therefore, be credited to an introduced nucleus. Briggs and King's nuclear transfer procedure involved first activating an oocyte at metaphase II of meiosis by pricking it with a glass needle, which initiated the metabolic changes normally induced by the sperm. Approximately 15 minutes later, a pit (black dot) appearing on the surface of the oocyte was microsurgically removed with another glass needle; this surgery removed the oocyte's chromosomes, resulting in an enucleated egg. Finally, a blastula cell was aspirated into a glass micropipette whose lumen was slightly smaller than the diameter of the cell to gently break the cell membrane. The broken cell was then transferred to the animal hemisphere of the enucleated egg, which permitted the nucleus to interact with the cytoplasmic molecular milieu of the oocyte and to undergo nuclear reprogramming (Figures 2 and 3). Nuclear transplantation in amphibians using embryonic cells as nuclear donors. Adult leopard frogs (A) are mated in the laboratory, and the resultant fertilized eggs (B) are permitted to develop to the blastula stage (C), with approximately 3000 cells. The blastula is dissociated to yield single cells (D) that will be used as nuclear donors. A gravid female (E) provides a recipient ovum which is by pricking with a glass The ovum is with another glass needle, as in this with a of a A dissociated cell (D) is into a micropipette and into the enucleated ovum When all of this are performed one or more cloned frogs are from and used with leopard frog Rana produced by nuclear transplantation In their the of the Briggs and King that developed from some enucleated eggs with blastula this stage, the the experiment because they were not in cloning animals but rather in nuclear when they early nuclei (Briggs and King they and that the into Briggs and King cells from blastula and early because (reviewed in Spemann had that when of embryos at were to other of embryos, the developed to the the cells of the transplanted were the procedure had not produced animals from nuclei of it could not have been applied to nuclei from determined and to study nuclear The of blastula nuclei was when blastula nuclei were found to the development of adult frogs that produced In the nuclei a in and in The resulting frogs the that the frogs were from nuclei and not oocyte nuclei from other in the United States, and the too the of blastula nuclei in other frog as as in some (reviewed in The of nuclear transplantation in a that the method of nuclear transplantation should be valuable principally for the study of nuclear differentiation, it may also have other (Briggs and King 1952, p. We this research and other of cloning In it is that the amphibian nuclear transplantation procedure became the for cloning animals. cells that are to rise to if in place if to a to rise to develop as the other cells that are to rise to into cells when in Thus, cells, not as cells, are determined to a (Spemann 1938). King and Briggs found that with embryonic more and more transplanted nuclei a of differentiation This was by other for several frog and (reviewed by made this was the that similar results were obtained by King and Briggs with and by with the is primitive and and it is more and of the and it more the between the animals, similar of nuclear potential was observed in the transplanted nuclei from (reviewed by Although cells of embryos have lost nuclear was to nuclear changes with to the transplantation procedure. the to complex and the to the cell of the oocyte Of the complete blastulae that developed from enucleated egg cells into which nuclei had been transplanted using developed into when the were not of the blastulae developed into years that differentiation the of then it be is as as nuclei from are The of is that in the nuclear transfer procedure the developmental of nuclei from developmental a of amphibian nuclei enucleated eggs to develop into one nucleus from from nuclei 2 nuclei and and 2 nuclei and et al. However, it is not known whether the nuclei from cells or from cells. In to the amphibian nuclei from no adult nuclei were found to be (reviewed by nuclear transfer of and adult cells from and the nuclei of cells to be but not nuclei from and into enucleated eggs the development of or (reviewed by The from nuclei from the formation of that for to a (Di et al. These are for several One is that the was by the shape and of the The cell type of nuclei in a cloning experiment is not known because of the of that are of and cells. there be no as to the cell type because of their and the of the amphibian cell is with a of the cell in this and the of of the study was that nuclei were first into metaphase oocytes and for one while the oocytes into metaphase II oocytes The oocytes were then by of a glass needle, and the nucleus was the nucleus in the cytoplasm. the blastulae had developed that then became nuclear for enucleated metaphase II These cloned embryos developed into with (Figure The of metaphase oocytes for the genome was on in which nuclei transferred to metaphase II oocytes to development of the beyond the early those nuclei exposed first to metaphase and then metaphase II oocyte cytoplasm the to develop into (Di and These experiments were on the hypothesis that molecular of the oocyte cytoplasm that oocyte to in would the of the developmental potential of nuclei was by this the molecular for this nuclear reprogramming from the transplantation of a nucleus. The nucleus was in oocyte cytoplasm and transplanted into an enucleated egg. The had and a functional the and it was to from et Finally, the experiments in the cloned animals produced with adult nuclei the of Dolly, the sheep cloned from an adult mammary gland cell by et al. the in the Dolly developed from a one in the part of the cell One but the of the with the results of the results of nuclei in the that nuclei more normally into the cell of the than those in the other cell the of that cells rise to more cells. This view that of cells be that the of all cells in a cells in a with is because a of do not in their between cells and cells When one of us joined the laboratory of King in it was that the cloning procedure could be used to determine whether the molecular of the oocyte and that of the embryo could the cell differentiation would in the embryo or produced from the transplantation of a cell nucleus into an enucleated egg, it would that of a genome could, rise to This in would support for the that differentiation be developed as a of for This then was the for the cloning of a The for cloning and was the et al. 1999) of pipiens. Although little was known the of cells to rise to cells by at the time more is known now and et al. or are for nuclear transplantation it was to that cells have a (Di et al. and The cell nuclei were to partial and complete in enucleated some of the complete blastulae developed into embryos and and and These that the nucleus the for embryonic and development. However, because development of nuclear from cells as the of the nuclear the nuclei were from adult it became to that the nuclear developed from the nucleus and not from an egg nucleus. it was to that development from a nucleus from a cell and not from one of the cells in the The was obtained with several the of the of the of the cells by their in with and the of cell in et al. The results to the that the genome could the development of early determine if more differentiation could be by the of nuclear transplant embryos were to of a and the were for the of et al. of all that were equivalent to the of of the were observed in was no of in any of the The reprogramming of cells is in the context of the of research known as differentiation The of differentiation in of the and induced differentiation of other cell the view that the of known as differentiation may in older of Cloning in were during the of the eggs was not several nuclei into the of each or fertilized egg at the cells, the of the cells, would later The to be that in some nuclear as as nuclei would the cells. In cases, the fertilized egg developed into The egg developed into nuclear that were by the cells of the embryos or the of the into of embryos developed into These were and produced that from containing or Thus, experiments the of the original transplanted and early Nuclear in were initiated by the in the early in and by One of their was to for and this they produced by a blastula nucleus of one into an enucleated oocyte from a of nuclei and cytoplasm from and in the of blastula to the original the and the of producing valuable by this Cloning of nuclear transfer was in mice during the early approximately years the first were Although there was in cloning to efforts were were for the in and in of oocytes and embryos. using cells were in the of nuclear during that was for cells of and In a of experiments blastomere separation and of and of cell cells into the had already that is in at least the stage (reviewed in In to to the of to on nuclear transfer to oocytes of animals. Nuclear in are performed by cell using a procedure similar to the one developed for nuclear and but with some (Figure A micropipette is the the the and the oocyte's the micropipette is then used to the chromosomes, and first a micropipette containing an cell is the and the cell is gently into the between the oocyte's and the The two cells are usually by the in the cell of the oocyte and cell, the of each to The some in cases, is in some nuclei may be into oocytes that are (Wakayama et al. 1998, Wakayama and Yanagimachi The nuclear are in various and then transferred to the of Nuclear transplantation in the embryonic cells for nuclei are obtained from an in egg. cytoplasm is by of an A nucleus is the of an enucleated The egg and its transferred cell are by the nucleus to interact with the recipient cytoplasm. The egg containing the transferred nucleus is in and transferred to a The of the that it from the that provides the recipient oocyte cytoplasm The cloned the of the nuclear not the of the recipient cytoplasm. from et at with has been for nuclei from various of and (reviewed by and et al. In all cases, the developed into and produced the cloned by nuclear transfer are two (Figure from embryos et al. We a on of their In some cases, were produced from a single will be valuable for testing on animals with an identical were in and et al. and in a of animals produced by cloning the cloning of et al. Wakayama et al. calves from one et al. et al. et al. and calves were produced by cloning and Finally, cloning of nuclei from the stage in and identical were cloned from cells the human for and one is producing the in et al. Although cloning of nuclei has been successful, the cloning from more as in a cloning for has been with and and cells, as as with adult sheep mammary and and cells. The of on the number of embryo transferred to are for from cells et al. for calves from cells et al. 1998), and for calves from cells et al. to the cloning from adult cells, the are for from mammary (Wilmut et al. for mice from cells (Wakayama et al. 1998), for mice from cells (Wakayama and Yanagimachi 1999), for calves from and cells (Kato et al. 1998), and for calves from cells et al. However, for those on the application of cloning to humans, we the throughout the procedure. The Dolly as we will began with to a mammary gland cell to an the development of this one ewe a of and the in during or during various and Although the for and cloning from adult cells are than for humans, the resulting is in sheep and further and cloned calves from cells of the cell of This result established the of cloning from cells, and it that be produced by cells with a and then using cells as cells for nuclear transfer. two have now produced cloned animals. et al. cloned of which containing the human for cells in as and were with of the for and human of the for The were then exposed to which all cells those those cells that had the genes for therefore, and were used for nuclear transfer. The of the the for human to be in a that in the mammary the from the will be used to (Wilmut calves were cloned from cells of a cell that contained a by a et al. In the be to a of complex human for human should be that the of animals by cloning is more than by into the of fertilized are several reasons for this the that cells be in nuclear transfer. Cloning experiments have provided valuable into a number of such as nuclear and genomic The was used in frog cloning to the and molecular changes in nuclei transplanted into oocyte cytoplasm. The cytoplasm of eggs transplanted nuclei to and The transplanted nuclei at later embryonic at the time as embryonic nuclei from fertilized eggs to This of nuclear also to genes (reviewed in during the first cell of frog nuclear move between the transplanted nucleus and the egg move from the cytoplasm into the nucleus (Di and and This result that the are techniques are to the of When nuclei were in from amphibian were by somatic and the molecular and was when the nuclei were in of amphibian somatic and were from into the egg and were into and somatic and were and to in nuclear transplant embryos of and that changes similar to those observed in amphibian nuclear also during nuclear reprogramming et al. The of nuclear reprogramming is by the that nuclear reprogramming or its in amphibian and nuclear and development (reviewed in are to the molecular changes involved in nuclear this of research may result in some of the of cloning to humans. if scientists could in molecular how a nucleus is it be possible to of could be removed from a and in the cell is could be to a type of cell differentiation (e.g., or the could be to the the cells would be recognized as and not Cloning experiments have the potential of a genome during cells in have a and However, even cloning cell nuclei of blastula cells were to development transplantation to even nuclei that had more than cell had the to the formation of et al. is that somatic nuclei are to some in oocytes because oocytes a of molecular that support nuclear and may the of which are normally in during the and may also be in animal Dolly and two other cloned sheep were to have that were than those of et al. their the cloned sheep were and remains to be if the will have an effect during the of the Nuclear transfer and molecular in mice genomic a that the differential of of nuclear of two or two to nuclear nuclei and et al. of embryos that genes are from and at in development Thus, an embryo with two genes to a is for this that the embryo a set of genes from the and the for development. The results in mice to the of the of several human such as and which but are to in of in and in of their in the (e.g., to in is but the for its in the of humans. other in was initiated to In to the of the nucleus during cell differentiation, the procedure also provided into of the nuclei by oocyte nuclear and genomic will continue to yield in and other scientists have in frog we are to see of research in the of cloned that will human for the of human Cloning will result in the of the of animal to study and human and of animal and for to humans. We consider of cloning they are the reasons Dolly was Dolly the news became with the of cloning human which stimulated a ethicists, theologians, of We do not to the and of we the reasons for our that human cloning is and We human cloning as the attempt to a human by any cloning blastomere of embryos, and nuclear Nuclear transplantation of or adult cells from all results in animals at a that with the of the The result from nuclear reprogramming and cell other may in some be nuclear from all development at various nuclear transfer and and In transplant embryos may to and those that do may at various embryonic and Finally, those nuclear that are may birth or with birth of nuclear transplant embryos from and molecular that could for the (reviewed in even some nuclear transplant frog blastulae from embryonic or adult nuclei contained in some of their cells, the in other cells of the blastulae would in nuclear for further and development. we consider the of any cell type those from for human cloning and In the of Dolly, she was the of of oocytes and cells that were from of mammary Even as cloning of adult nuclei more there will be to humans. the cells could in from during the of the donor. could also in the cells during cell an that is not are other in the cell the of the and in oocytes from be with the Finally, it is to consider that which another for and should not be in of all of we human cloning. of and provided on an early of this of of provided valuable in several of the for of the of some were not and we for research on nuclear transplantation of cells was by from the for cloning research on the genomic potential of frog cells was by from the United National Institutes of Health and the National Science