aSome tadpoles advanced beyond the feeding stage. fcSome survived for 1 mo.

aSome tadpoles advanced beyond the feeding stage. fcSome survived for 1 mo.

that the percentage of transplants developing had to significantly exceed the percentage of undifferentiated cells. Those cases, including one of the criteria, are listed in Table 2 (14,64,69-73) and described briefly here.

The donor cell types directly identified under the stereomicroscope at the time of nuclear transfer were melanophores, erythrocytes, erythroblasts, and leukocytes. The unique properties of melanophores and erythroid cells, melanin pigment, and hemoglobin, respectively, permitted direct identification. The leukocytes, separated from erythroid cells via centrifugation of whole blood, were taken from the buffy coat layer and verified microscopically to lack hemoglobin at the time of nuclear transfer. In two other studies using skin cells and lymphocytes, the donor cells were derived from a nearly homogeneous population. Correlative studies on skin cultures revealed that more than 99% of the cells were producing immunoreactive keratin and correlative studies on lymphocytes showed that 96.1-98.7% of the cells were producing immunoglobulins.

Nuclei from six differentiated somatic cell types directed the formation of prehatching tadpoles and nuclei from five specialized cell types programmed for the development of posthatching tadpoles. Tadpole development was most extensive when Rana erythrocyte, erythroblast, and leukocyte nuclei were injected into metaphase I oocytes than when nuclei were injected into diplotene or metaphase II oocytes. The greatest percentage and most advanced tadpoles emerged from Rana erythrocyte nuclei of juvenile frogs that were tested in oocytes at first meiotic metaphase (14). In that study, 7.8% attained the feeding stage or beyond. Three original nuclei after recloning gave rise to 19 clonal tadpoles. All 19 tadpoles fed, differentiated hind limb buds, and in the best cases, survived as independent organisms up to 1 mo. Metaphase I oocytes were used for two reasons: to induce breakdown of the nuclear membrane so that essential cytoplasmic molecules could access the chromatin and to provide more time for nuclear reprogramming of the G0 erythrocyte nucleus. After injection of the nucleus, the nuclear envelope disappeared and the interphase nucleus converted into metaphase chromosomes aligned on a newly induced spindle. Approximately 24 h later, the oocyte matured, was activated, and the egg nucleus was removed. However, when erythrocyte nuclei were tested in metaphase II oocytes, they were forced into the first mitotic metaphase about three hours after injection, had little time to under reprogramming, and developed no further than the early gastrula stage (73). Recently, nuclear envelope permeabilization was shown to optimize chromatin remodeling in one-cell stage mammalian clones (74). Although the use of Rana metaphase I hosts led to enhanced expression of developmental potential in amphibians, at least for ery-throid and leukocytic nuclei, conditions were not found in amphibian cloning to reveal totipotency in adult cells. We had to wait for progress in mammalian cloning where "the long cell cycles of cleavage stages in the mammalian embryo may permit more extensive molecular reprogramming of adult nuclei and better integration into the host's cell cycle than that achieved with amphibian nuclear transplants, in which the cell cycles of the host are very rapid" (p. 213 [5]).

7. Clarifications From Mouse Cloning

Recent murine studies clarified two unresolved questions in frog cloning: the totipotency of fully differentiated somatic cells and the phenotypic restrictions of some transplants from endodermal and neural nuclei.

Throughout the decades of frog cloning, there was only one claim for the totipotency of nuclei from fully differentiated somatic cells, namely from intestinal cells of Xenopus larvae (11). In that study, 4 (0.6%) adults, two of whom were fertile, developed from four original nuclei. Recently, because of the regular presence of less differentiated stem cells intermingled within differentiated tissues, Hochedlinger and Jaenisch (20,75) tested the nuclear potential of murine Band T-lymphocytes under conditions that would yield unequivocal results. Mature lymphocyte nuclei carrying fully rearranged immunoglobulin or T-cell receptor alleles from cell lines derived from the peripheral lymph node were transferred into enucleated oocytes and embryonic stem (ES) cell lines were derived from the subsequent blastocysts. ES cells were then injected into tetraploid blastocysts, a procedure in which the embryo develops from the ES cells and the extraembry-onic tissues from the tetraploid host (76). Sixteen fertile adults cloned from B-cell nuclei carried the fully rearranged immunoglobulin alleles in all tissues, and a nonviable neonate cloned from a T-cell nucleus carried the rearranged T-cell receptor genes in all tissues. The production of monoclonal mice with the genetic markers of the donor nuclei demonstrated unequivocally that terminally differentiated somatic cells could be reprogrammed to produce clones. The authors critically point out that the two-step procedure does not show that nuclei of B- and T-cell lymphocytes can be directly reprogrammed to direct the formation of extraembryonic cell lineages, but in the past, ES cell nuclei have been shown to specify the development of both embryonic and extra-embryonic tissues (77).

Five decades ago, a characteristic syndrome of morphological and cellular abnormalities was reported in some Rana nuclear transplants derived from endodermal nuclei of late gastrulae and neurulae (62). During postneurula and early tadpole stages, some clones displayed deficiencies and degenerative changes primarily in the ectodermal and mesodermal derivatives, but good differentiation in the endodermal derivatives, a phenotype termed the endoderm syndrome. A second group of abnormal clones displayed generalized deficiencies, whereas the third group was normal. Both the endoderm syndrome and the generalized deficiencies were stable as neither was reversed by serial transplantation (62,78). Furthermore, the restrictions were intrinsic; they could not be reversed by parabiosis of nuclear transplant embryos with normal ones (79), nor were they corrected when the haploid nucleus of the egg was combined with the diploid endodermal nucleus (80).

Clarification of the three different phenotypes was provided primarily from chromosomes studies of the nuclear transplants (81). Those with generalized deficiencies displayed extensive and variable aneuploidy, with severe structural alterations in the chromosomes, whereas the group displaying the endoderm syndrome (40%) was euploid or predominantly euploid. The third group that developed normally showed no chromosomal abnormalities. These studies were confirmed and extended to include chromosomal analysis of clonal donors used in serial nuclear transplantation (78). Seventy-five percent of the abnormal clones that were euploid or predominantly euploid exhibited the endoderm syndrome, whereas the remaining abnormal ones displayed generalized cellular deficiencies and were extensively and variably aneuploid with structural alterations in the chromosomes.

An analogous study on clones from nuclei of the presumptive medullary plate region of late gastrulae and the definitive medullary plate of early and mid-neu-rulae from the ectodermal lineage revealed different but complementary restrictions (81). In addition to normal larvae, there were two groups of abnormal clones during postneurula and larval stage stages: 13% displayed cellular deficiencies mainly in mesodermal and endodermal derivatives but good differentiation in the organs and tissues of ectodermal origin. The chromosomes in half of this group were examined during larval stages and found to be euploid with an apparent normal karyotype. The other group comprising abnormal postneurulae and larvae (87%) exhibited generalized cellular deficiencies and the chromosomes analyzed from a sample were abnormal in number and structure.

The two distinct but complementary phenotypes suggested that some endodermal and neural nuclei acquire stable properties for specific pathways of differentiation; however, the data were limited because clones with numerical and structural chromosome abnormalities had to be eliminated and, furthermore, submicroscopic aberrations, if present, could not have been detected. Later, analysis of nuclear transplants during the first cell cycle revealed the chromosome abnormalities were traceable to an asynchrony in the cell cycles of the donor nuclei and recipient oocytes (82,83).

Although these phenotypes were described long ago, the genetic mechanism responsible for them remained elusive, although we considered different patterns of gene expression probably accounted for the phenotypes. The restrictions in Rana nuclear transplants have now been clarified in murine clones (75,84). Global gene expression of more than 10,000 genes was assessed by microarray analysis of RNA from the placentas and livers of neonatal cloned mice derived from either cultured embryonic stem cells or freshly isolated cumulus cells. Although most abnormally expressed genes were common to both types of clones, a smaller set of abnormally expressed genes differed between the ES and cumulus cell-derived clones, reflecting the particular donor nucleus. Other murine studies have revealed altered phenotypes in clones that are donor specific as early as preimplantation stages (85).

The problem of the endoderm/neural syndromes in frog clones is a good paradigm for young scientists: record what you observe and eventually its significance will be revealed. The lengthy cell cycle (approx 1 d) in mammalian one-cell embryos offers a distinct advantage over amphibian cloning where the first cell cycle is quite short (approx 1 h in Xenopus and 3 h in Rana), causing an asyn-chrony between the cell cycle of the donor nucleus and the oocyte that induces chromosome abnormalities. Although chromosome aberrations can occur in mammalian cloning (e.g., ref. 86), they are less frequent than that observed in amphibian clones. However, synchrony of the cell cycle is not sufficient. Another major problem in both mammalian and frog cloning is the incomplete reprogramming of gene expression in donor nuclei. Perhaps this problem may be solved in mammals for the nonimprinted genes by exposure of donor nuclei in vitro to appropriate inducers before nuclear transfer. But how to reset the correct pattern of genetic imprinting that is set over a protracted period from germ cell differentiation through embryogenesis will be a more formidable task.

8. Nuclear Reprogramming

Nuclear reprogramming, a phrase used early in amphibian cloning, denoted the morphological and molecular changes that nuclei undergo after transplantation into oocyte or egg cytoplasm and, later, included changes in chromatin and gene expression. Probably one of the most significant byproducts of nuclear transfer will be the understanding of the molecular mechanisms controlling the reversible expression of chromatin and genes in transplanted nuclei. In the immediate future, such knowledge should improve cloning efficiency. In the more distant future, it might permit us to convert differentiated adult cells into immature cells and then redirect the latter into specific cell types required for tissue repair. Here, I consider briefly some examples of nuclear reprogramming in transplanted amphibian nuclei, but first I cite two pioneering studies demonstrating the cytoplasmic control of nuclear and chromosomal behavior. More than 80 yr ago, Brachet (87) observed that, when sperm prematurely entered sea urchin oocytes still undergoing maturation divisions, the sperm chromatin rapidly condensed into chromosomes similar to those of the oocytes; the same phenomenon was later observed in precociously inseminated oocytes of amphibians by Battaillon and Tchou-Su (88).

Initial observations of amphibian embryonic nuclei transplanted to metaphase II oocytes revealed morphological changes similar to those the egg and sperm nuclei undergo following fertilization: nuclear enlargement, chro-matin decondensation, nuclear migration toward the equator, and spindle formation for the first mitosis (89,90). Numerous studies ensued in which nuclei from various cells types were injected into oocytes or eggs of Buffo, Pleurodeles, Rana, and Xenopus that firmly established the cytoplasmic control of nuclear, chromosomal, and gene function (5,49).

In the diplotene host, transplanted nuclei enlarged, transcribed RNA, and synthesized new proteins. Liver nuclei of A. mexicanum directing the synthesis of alcohol dehydrogenase, a liver-specific enzyme, and lactate dehydroge-nase, common to many cell types, were injected into diplotene oocytes of A. mexicanum. Starch gel electrophoresis differentiated the enzyme forms between the two species and revealed the oocytes ceased synthesizing alcohol dehydrogenase but continued making lactate dehydrogenase (91). Likewise, when Xenopus nuclei from a kidney cell line were injected into Pleurodeles oocytes, two-dimensional gel electrophoresis revealed cessation of Xenopus kidney-specific proteins and expression of Xenopus oocyte-specific proteins (92). Also, when nuclei from Xenopus erythrocytes and cultured kidney cells were injected into diplotene oocytes, oocyte-specific 5s ribosomal genes were activated (93).

After germinal vesicle breakdown, transplanted nuclei from various cell types converted into metaphase chromosomes aligned on newly induced spindles in concert with the oocyte's nucleus (p. 97 [5]). When nuclei were injected into diplotene or maturing oocytes and, subsequently, the oocytes were matured and activated, the nuclei transformed into pronuclei (94,95).

In activated eggs transplanted nuclei were induced to synthesize DNA and enter mitosis (96). During the first cell cycle, a bidirectional exchange of proteins occurred between injected nuclei and the host cytoplasm. Transplanted nuclei accumulated radioactive labeled proteins of the cytoplasm (97,98) that were composed of histone and nonhistone proteins (99). On the other hand, when labeled nuclei were injected into activated eggs, histone proteins were primarily retained whereas a significant amount of nonhistone proteins were lost (100,101). Two decades later, the remodeling of chromatin was directly analyzed. After transcriptionally inactive sperm nuclei were incubated in extracts from activated amphibian eggs, sperm-specific histone proteins were replaced by somatic histones H2A and H2B via the molecular chaperon nucle-oplasmin (102). Similarly, erythrocyte chromatin was remodeled when their nuclei were incubated in extracts of activated amphibian eggs: somatic histones H1 and H10 were released from chromatin into egg cytoplasm, oocyte-specific linker B4 and high-mobility group 1 were incorporated into remodeled chro-matin, and somatic histones H2A and H4 were phosphorylated (103).

The switching of gene expression was examined in Xenopus eggs and embryos. For example, nuclei synthesizing 28s and 18s ribosomal RNA or muscle actin RNA ceased to do so after nuclear transfer, but began at the gastrula stage when controls did (49).

Many of the aforementioned studies have been confirmed in mammalian clones and significantly extended, especially deciphering various molecular, biochemical and physiological changes occurring during nuclear reprogramming (see Section 3.).

9. Nuclear Transfers in Insects, Ascidians, and Fish

In addition to the cloning of various mammalian species, the only other metazoan animal groups to be cloned have been insects and fish. In the case of ascidians, nuclear transplantation was used to study the effect of cytoplasm on nuclear function but mature clones were not produced.

Cloning studies in Drosophila were launched during the 1960s. Because enu-cleation of the fragile host eggs was not feasible, investigators injected several genetically marked nuclei into the posterior region of each unfertilized (12) or fertilized (13) egg at the site where pole cells, the progenitors of the germ cells, would later form. They assumed correctly that, in some nuclear transplants, host as well as injected nuclei would populate the pole cells and, subsequently, the fertility of adult gametes from both host and injected nuclei could be assessed. In many cases, the fertilized egg hosts developed directly into normal adults. In the case of the unfertilized but activated egg hosts, they formed defective nuclear transplants but were rescued by transplanting the pole cells of embryos or gonads of larvae into normal hosts. When the adults successfully mated, they produced normal progeny from both the host and donor nuclei, demonstrating the totipo-tency of the preblastoderm (13) and early gastrula (12) donor nuclei. The impetus for nuclear transfers in Drosophila in the 1960s was the expectation that the valuable genetics available in this species would be helpful in analyzing problems of nuclear differentiation but, unfortunately, cloning in this species has not continued. Perhaps important problems can still be pursued. For example, insect clones derived from donor cells containing modified genomes might alleviate the negative effects of insects on fauna and flora in general and especially on humans.

In the protochordate ascidian, Ciona intestinalis, no clones were produced, but the effects of cytoplasm from mosaic eggs and embryos on transplanted nuclei were studied in intraspecies (104) and interphylum (105) combinations. Interspecific combinations comprised nuclei from ectodermal, mesodermal, or endodermal cells of gastrula or tailbud embryos of one species that were injected into non-nucleated egg fragments from another species. Cytological examination of the resultant embryos revealed that nuclei from all sources participated in the formation of cells in all three germ layers. They concluded that cell differentiation was principally determined by the components of the mature egg cytoplasm, and nuclear function was influenced by components in the surrounding cytoplasm (104).

Interphylum nucleocytoplasmic hybrids, containing a blastula nucleus from the sand dollar Echinarachnius parma and an activated and anucleated egg fragment of C. intestinalis, were analyzed to determine whether cytoplasmic determinants from Chordata would still affect a nucleus from the phylum Echinodermata. Despite the distance between the phyla, the ascidian cytoplasm influenced the supermolecular assemblage of the components of extracellular matrix and neural cells (105).

Nuclear transplantation in fish was initiated by the late T. C. Tung (Tong Dizhou) in the early 1960s in China and extensively investigated by Yan (8). One of Yan's goals was the production of fish clones for agricultural and commercial purposes. To this end, he constructed nucleocytoplasmic hybrids by transplanting a blastula nucleus of one species into an enucleated oocyte from a different species. Combinations of nuclei and cytoplasm from different genera and even different subfamilies resulted in fertile adults, demonstrating the totipotency of fish blastula nuclei and their compatibility with the cytoplasm from another genus and even from another subfamily. The intergenera constructs exhibited higher growth rates, increased protein content, and lower fat content than the original species, suggesting the feasibility of producing commercially valuable fish by cloning.

Recently, Wakamatsu et al. (106) in Japan initiated a cloning program with embryos of the small laboratory medaka fish, Oryzias latipes to study nuclear differentiation and gene function. Nuclei from blastula cells containing the gene for green fluorescent protein (GFP) were injected in unfertilized eggs enucleated by

X-ray irradiation. Six fertile adults resulted expressing GFP that they transmitted to the F1 and F2 offspring in Mendelian fashion. Also, fertile zebrafish expressing and transmitting GFP to their progeny were produced from embryonic fibroblast nuclei (107). The fish model should be valuable for cell lineage studies, especially in those species whose internal development is somewhat transparent.

Throughout this review my use the of the word clone(s) has conformed to the current meaning adopted by scientists and nonscientists alike, namely an organism derived from an oocyte injected with any cell nucleus. Because a metazoan clone may not share in its oocyte the same mitochondrial DNA and maternal RNAs originally endowed to the donor cell, classifying a nuclear transplant as a clone is not scientifically correct, but this appellation has gained extensive use. Now that the word animal cloning has become ubiquitous for one meaning, it is appropriate that a recent scholarly report on the origin and evolution of the many meanings of clone has been published (108).

10. Concluding Comments

Just more than 50 yr ago, the first tadpoles were cloned from frog blastula nuclei (1). It is a challenge in this small review to capture fully the significant events that occurred during the past 50 yr, but I have attempted to highlight the historical roots of nuclear transfer, as well as the progress made in cloning non-mammalian species. In succeeding chapters experts in mammalian species will discuss advances in mammalian cloning, cell reprogramming, and transgenesis. As one of the early frog cloners and an enthusiastic observer of mammalian cloning, I would like to briefly cite four advances in mammalian cloning that I consider highly significant: (1) successful cloning of adult cells; (2) unequivocal demonstration that nuclei from fully differentiated cells can direct the formation of fertile adults; (3) important advancements in our knowledge of nuclear reprogramming; and (4) the construction of transgenic clones, especially via gene targeting of donor cells. I look forward to continued advances: the production of animal clones with modified genomes for commercial improvements and resistance to diseases as well as for medical applications to humans and, on the cellular level, further advances in nuclear reprogramming and, hopefully, to the extent that we may someday control the state of cell differentiation in vitro for applications in vivo.


I thank L. D. Etkin and K. Latham for their constructive comments on a previous draft of the manuscript and Barbara Engle for preparing the figures. The author's research was supported by grants from The National Science Foundation and the National Institute of General Medical Sciences of the United States.


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