核移植与供体细胞

“多莉”羊的诞生是生物界的一个里程碑,它之所以引起如此大的轰动主要是因为它来源于培养的成年绵羊乳腺上皮细胞,这是人类第一次证明分化的体细胞可以被重编程后恢复全能性并最终分化发育成一个动物个体。这说明哺乳动物分化的体细胞核仍具有全套的遗传物质并能够被卵母细胞逆转恢复全能性。然而,关于多莉的供体细胞来源却一直是克隆领域的一个谜。由于体细胞克隆的效率非常低,而用于核移植的供体细胞悬液中往往含有多种类型的细胞,这使得我们很难确切地知道最终获得的克隆动物是来源于哪一种细胞。这种不确定性给我们研究核移植诱导体细胞重编程的机制带来了很大的困难,因此,对供体细胞的研究也是核移植研究领域的一个重要课题,这包括各种组织来源的体细胞是否均可以用于核移植,终末分化的体细胞是否能够用于核移植,组织干细胞是否更有利于体细胞重编程,供体细胞的分化状态是否与核移植的效率有关,死亡的体细胞是否也可以用于核移植等等。本文综述了核移植中与供体细胞相关的最新研究进展。     

Remodeling of Centrosomes in Intraspecies and Interspecies Nuclear Transfer Porcine Embryos

Remodeling of donor cell centrosomes and the centrosome-associated cytoskeleton is crucially important for nuclear cloning as centrosomes are the main microtubule organizing centers that play a significant role in cell division and embryo development. Centrosome dysfunctions have been implicated in various diseases including cancer and metabolic disorders and may also play a role in developmental abnormalities that are frequently seen in cloned animals. In the present studies we investigated microtubule organization and the reorganization and fate of the integral centrosome protein γ-tubulin and the centrosome-associated protein centrin in intraspecies (pig oocytes; pig fetal fibroblast cells) and interspecies (pig oocytes; mouse fibroblast cells) reconstructed embryos by using antibodies to γ-tubulin or GFP-centrin transfected mouse fibroblasts as donor cells. Microtubules were stained with antibodies to α-tubulin. In-vitro-fertilized oocytes and nuclear transfer (NT) reconstructed oocytes were sequentially analyzed at different developmental stages. Epi-fluorescence results revealed mitotic spindle abnormalities in NT embryos during the first cell cycle (39.4%, 13/33) which were significantly higher than those in IVF embryos (17.0%, 7/41). The abnormalities in IVF embryos are due to polyspermy while the abnormalities in NT embryos are due to donor cell centrosome dysfunctions. In the NT embryos with abnormal microtubule and centrosome organization, γ-tubulin staining revealed multipolar centrosome foci while DAPI staining showed misalignment of chromosomes. In intraspecies and interspecies embryos the GFP-centrin signal was detected until 3 hrs after fusion. GFP-centrin was not detected at 8 hrs after NT which is consistent with previous results using anti-centrin antibody staining in intraspecies NT porcine embryos. These data indicate that 1) abnormalities in microtubule and centrosome organization are associated with nuclear cloning at a higher rate than observed in IVF embryos; 2) centrosome and cytoskeletal abnormalities in IVF embryos are due to polyspermy while centrosome and cytoskeletal abnormalities in NT embryos are due to donor cell centrosome dysfunctions; and 3) GFP-centrin of the donor cell centrosome provides a reliable marker to follow its fate in intraspecies reconstructed embryos.     

In vitro development of nuclear transfer embryos derived from porcine embryonic germ cells and their descendent neural precursor cells

Undifferentiated stem cells may support a greater development of cloned embryos compared with differentiated cell types due to their ease of reprogramming during the nuclear transfer (NT) process. Hence, stem cells may be more suitable as nuclear donor cells for NT procedures than are somatic cells. Embryonic germ (EG) cells are undifferentiated stem cells that are isolated from cultured primordial germ cells (PGC) and can differentiate into several cell types. In this study, the in vitro development of NT embryos using porcine EG cells and their derivative neural precursor (NP) cells was investigated, thus eliminating any variation in genetic differences. The rates of fusion did not differ between NT embryos from EG and NP cells; however, the rate of cleavage in NT embryos derived from EG cells was significantly higher (p < 0.05) than that from NP cells (141/247 [57.1%] vs. 105/228 [46.1%]). Similarly, the rate of blastocyst development was significantly higher (P < 0.05) in NT using EG cells than the rate using NP cells (43/247 [17.4%] vs. 18/228 [7.9%]). The results obtained from the present study in pigs demonstrate a reduced capability for nuclear donor cells to be reprogrammed following the differentiation of porcine EG cells. Undifferentiated EG cells may be more amenable to reprogramming after reconstruction compared with differentiated somatic cells.     

Assessment of chromosomal abnormalities in bovine nuclear transfer embryos and in their donor cells

Chromosomal anomalies were assessed in nuclear transfer (NT) embryos (n = 148) at 1-4-cell stage (n = 88), and morula (n = 60), as well as in donor cells (n = 97) derived from two different cell lines. Two different cytogenetic approaches were used: conventional karyotyping and fluorescent in situ hybridization (FISH) with painting probes, specific for bovine X and Y chromosomes. The total rate of NT embryos with abnormal nuclei was 43%. These anomalies were mainly nuclear fragmentation (30%), hypoploidy/hypoploidy-mixoploidy (9%, n = 14) and hyperploidy/hyperploidy-mixoploidy (3%, n = 5). The incidence at which these anomalies occurred in NT embryos varied according to the donor cell culture and paralleled the frequency of anomalies in donor cells. A higher frequency of total anomalies was observed in NT embryos (55%) derived from the donor cell cultures with the highest incidence of anomalies (23%). An increase in the rate of total anomalies of the cell, after transfer to recipient cytoplasm, was also observed. These results suggest that proper screening of donor cells for chromosomal anomalies must be performed prior to NT procedure. They also suggest that the NT procedure itself might have a detrimental effect on some mechanism of chromosome segregation and distribution during cell division.     

Production of transgenic bovine embryos by transfer of transfected granulosa cells into enucleated oocytes

Adult granulosa donor cells used in the nuclear transfer (NT) procedure can result in cloned cattle. Subsequently, it may be possible to use the same cell type to produce cloned transgenic cattle. Therefore, this study examined the effect of genetic manipulation and serum levels in culture of donor granulosa cells on developmental rates and cell number of bovine NT embryos. A primary cell line was established from granulosa cells collected by aspirating ovarian follicles. Cells transfected with a plasmid containing the enhanced green fluorescence protein (EGFP) gene, and non-transfected cells were used for cloning between passage 10 and 15 as serum-starved and serum-fed donor cells. There were no significant differences (P > 0.1) in cleavage rates or development to the blastocyst stage for NT embryos from transfected (60.4 and 13.5%, respectively) or non-transfected (61.9 and 14.1%, respectively) and serum-starved (60.6 and 13.4%, respectively) or serum-fed (61.3 and 14%, respectively) cells. Development rates to blastocyst stage of embryos produced using cells at passage 15 (27.1%) were significantly higher than those produced with cells at passage 10,11, and 13 (7, 11.5, and 14%, respectively, P < 0.05). Green fluorescence was observed at different intensity levels in all blastocyst stage embryos resulting from transfected donor cells. The results of the present study indicated that genetically modified granulosa cells can be used to produce transgenic NT embryos and primary transgenic adult cells at late passage may be more effective donor cells than earlier passaged cells.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.     

In vitro development of bovine nuclear transfer embryos from transgenic clonal lines of adult and fetal fibroblast cells of the same genotype

This study examined bovine cloning strategies that may be used for gene targeting in animals of known phenotypic traits. Fibroblast cells derived from an adult and a fetus of the same genotype were transfected with a plasmid (pEGFP-N1) containing the enhanced green fluorescence protein and neomycin-resistant genes. After transfecting 2 x 10(5) cells, 49 adult and 35 fetal cell colonies were obtained. Green fluorescence expression was observed in 35 out of 49 (71.4%) adult clones and in 30 out of 35 (85.7%) fetal clones. Developmental rates to the blastocyst stage following nuclear transfer (NT) did not differ among nontransfected cell lines (adult, 20.0%; NT fetal, 18.3%), whereas developmental rates were significantly lower for adult and fetal cell lines expressing enhanced green fluorescent protein (EGFP; 11.3% and 6.4%, respectively, P < 0.05). However, there was no decrease in NT developmental rates (19.8%) when donor nuclei from EGFP-transfected cell lines not expressing EGFP but retaining neomycin-resistant gene expression were used as donor nuclei. NT embryos from adult and fetal cell lines had similar morphology, cell number, and ploidy. The results indicated that adult and NT fetal cells (identical genotype) can complete clonal propagation, including transfection and selection, and can be used to produce transgenic NT embryos; however, a possible deleterious effect of EGFP on embryo development should be considered in future gene targeting studies.     

Donor cell differentiation,reprogramming, and cloning efficiency: Elusive or illusive correlation?

Compared to other assisted reproductive technologies, mammalian nuclear transfer (NT) cloning is inefficient in generating viable offspring. It has been postulated that nuclear reprogramming and cloning efficiency can be increased by choosing less differentiated cell types as nuclear donors. This hypothesis is mainly supported by comparative mouse cloning experiments using early blastomeres, embryonic stem (ES) cells, and terminally differentiated somatic donor cells. We have re-evaluated these comparisons, taking into account different NT procedures, the use of donor cells from different genetic backgrounds, sex, cell cycle stages, and the lack of robust statistical significance when post-blastocyst development is compared. We argue that while the reprogrammability of early blastomeres appears to be much higher than that of somatic cells, it has so far not been conclusively determined whether differentiation status affects cloning efficiency within somatic donor cell lineages.     

Nuclear transfer in cats and its application

Nuclear transfer (NT) technology is typically used for generating identical individuals, but it is also a powerful resource for understanding the cellular and molecular aspects of nuclear reprogramming. Most recently, the procedure has been used in humans for producing patient-specific embryonic stem cells. The successful application of NT in cats was demonstrated by the birth of domestic and non-domestic cloned kittens at a similar level of efficiency to that reported for other mammalian species. In cats, it has been demonstrated that either in vivo or in vitro matured oocytes can be used as donor cytoplasts. The length of in vitro oocyte maturation affects in vitro development of reconstructed embryos, and oocytes matured in vitro for shorter periods of time are the preferred source of donor cytoplasts. For NT, cat somatic cells can be synchronized into the G0/G1 phase of the cell cycle by using different methods of cell synchronization without affecting the frequency of in vitro development of cloned embryos. Also, embryo development to the blastocyst stage in vitro is not influenced by cell type, but the effect of cell type on the percentage of normal offspring produced requires evaluation. Inter-species NT has potential application for preserving endangered felids, as live offspring of male and female African wildcats (AWC, Felis silvestris lybica) have been born and pregnancies have been produced after transferring black-footed cat (Felis nigripes) cloned embryos into domestic cat (Felis silvestris catus) recipients. Also, successful in vitro embryo development to the blastocyst stage has been achieved after inter-generic NT of somatic cells of non-domestic felids into domestic cat oocytes, but no viable progeny have been obtained. Thus, while cat cytoplasm induces early nuclear remodeling of cell nuclei from a different genus, the high incidence of early embryo developmental arrest may be caused by abnormal nuclear reprogramming. Fetal resorption and abortions were frequently observed at various stages of pregnancy after transfer of AWC cloned embryos into domestic cat recipients. Abnormalities, such as abdominal organ exteriorization and respiratory failure and septicemia were the main causes of death in neonatal cloned kittens. Nonetheless, several live domestic and AWC cloned kittens have been born that are seemingly normal and healthy. It is important to continue evaluating these animals throughout their lives and to examine their capability for natural reproduction.     

Production of cloned pigs by nuclear transfer of preadipocytes established from adult mature adipocytes

The aim of the present study was to determine whether porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (SCNT) in pigs. Primary culture of porcine preadipocytes was established by de-differentiating mature fat cells taken from an adult pig. The cell cycle of the preadipocytes could be synchronized by serum starvation for 1 day, with a higher efficiency than control fetal fibroblasts. Incidence of premature chromosome condensation following nuclear transfer (NT) of preadipocytes was as high as that observed after NT with fetal fibroblasts. In vitro developmental rate of the NT embryos reconstructed with preadipocyte was equivalent to that of the fetal fibroblast derived embryos. Transfer of 732 NT embryos with preadipocytes to five recipients gave rise to five cloned piglets. These data demonstrate that preadipocyites collected from an adult pig are promising nuclear donor cells for pig cloning.     

Embryos derived from porcine skin-derived stem cells exhibit enhanced preimplantation development

Ongoing research to identify the most suitable type of donor cell for nuclear transfer (NT) has suggested that less differentiated stem cells may be better donors than other somatic cell types. Recently, we have reported the isolation and characterization of porcine skin-originated sphere (PSOS) stem cells from fetal skin, making it possible to test this hypothesis in a nonrodent animal model. In the present study, we have investigated and compared the feasibility and preimplantation developmental efficiency of using fetal PSOS cells and fibroblasts as nuclear-transfer donors. The majority of fetal PSOS cells are in the G1/ G0 stage of the cell cycle, which is desirable for NT. During long-term in vitro culture, fetal PSOS cells had greater genome stability, with a lower frequency of abnormal karyotypes than fetal fibroblast cells. Embryos cloned from PSOS cells showed enhanced preimplantation development compared with fibroblast cloned embryos, which is indicated by an increased rate of blastocyst development and a higher total cell number in Day 7 blastocysts. The gene expression profile of genes critical for early development from eight-cell-stage PSOS NT embryos more closely resembled the pattern observed from in vivo-produced embryos compared with that of fibroblast-cloned embryos. Cumulatively, our data suggest that fetal PSOS cells may be better donor cells for NT in the pig.     

Production of transgenic recloned piglets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer

We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.     

Production of transgenic recloned piglets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer

We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

Genetically modified animals have many poten-tial applications in basic research, human medicine and agriculture. Pronuclear DNA microinjection has been almost the only practical means of producing transgenic animals during the last 20 years, but the low efficiency (1%—5%)[1] of this method has actu-ally been the obstacle that hampered its further appli-cation in animal biotechnology. The birth of Dolly[2], the first somatically cloned animal, made it possible to produce transgenic animals b…     

Production of transgenic blastocyst by nuclear transfer from different types of somatic cells in cattle

The present study examined the effects of genetic manipulation to the donor cell and different types of transgenic donor cells on developmental potential of bovine nuclear transfer (NT) embryos. Four types of bovine somatic cells, including granulosa cells, fetal fibroblasts, fetal oviduct epithelial cells and fetal ovary epithelial cells, were transfected with a plasmid (pCE-EGFP-Ires-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant (Neor) genes by electroporation. After 14 days selection with 800 μg/mL G418, transgenic cell lines from each type of somatic cells were obtained. Nontransgenic granulosa cells and all 4 types of transgenic somatic cells were used as nuclear donor to produce transgenic embryos by NT. There was no significant difference in development rates to the blastocyst stage for NT embryos from transgenic and nontransgenic granulosa cells (44.6% and 42.8%, respectively), and transfer of NT embryos derived from transgenic and nontransgenic granulosa cells to recipients resulted in similar pregnancy rates on day 90 (19% and 25%, respectively). The development rates to the blastocyst stage of NT embryos were significantly different among different types of transgenic donor cells (P<0.05). Blastocyst rates from fetal oviduct epithelial cell and granulosa cell (49.1% and 44.6%, respectively) were higher than those from fetal fibroblast (32.7%) and fetal ovary epithelial cell (22.5%). These results suggest that (i) genetic manipulation to donor cells has no negative effect on in vitro and early in vivo developmental competence of bovine NT embryos and (ii) granulosa and fetal oviduct epithelial cells can be used to produce transgenic bovine NT embryos more efficiently. In addition, GFP can be used to select transgenic NT embryos as a non-invasive selective marker.     

Preimplantation and postimplantation development of rat embryos cloned with cumulus cells and fibroblasts

In this report we demonstrate the successful in vitro culture of fertilised embryos from 1-cell to blastocyst stage, albeit in a strain-dependent fashion. We report procedures for the enucleation of rat oocytes; nuclear transfer by injection of nuclei (NT) from adult rat cumulus cells, rat primary embryonic fibroblasts and genetically modified rat fibroblasts; and activation resulting in advanced preimplantation development. Blastocyst stage rat embryos were obtained after in vitro culture of nuclear transfer zygotes at similar frequencies with each of these nuclear donor cell types. Transfer of NT embryos to surrogate mothers leads to implantation of 24% of the zygotes. These results suggest that the nuclei of cultured rat cells, even following genetic modification, can be reprogrammed to support early embryonic development, which is a prerequisite to cloning the rat.     

Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.     

Development of interspecies cloned embryos in yak and dog

Interspecies nuclear transfer (NT) could be an alternative to replicate animals when supply of recipient oocytes is limited or in vitro embryo production systems are incomplete. In the present study, embryonic development was assessed following interspecies NT of donor cumulus cells derived from yak and dog into the recipient ooplasm of domestic cow. The percentages of fusion and subsequent embryo development to the eight-cell stage of interspecies NT embryos were comparable to those of intraspecies NT embryos (cow-cow NT embryos). The percentage of development to blastocysts was significantly lower (p < 0.05) in yak-cow NT embryos than that in cow-cow NT embryos (10.9% vs. 39.8%). In dog-cow NT embryos, only one embryo (0.4%) developed to the blastocyst stage. These results indicate that interspecies NT embryos possess equally developmental competence to the eight-cell stage as intraspecies NT embryos, but the development to blastocysts is very low when dog somatic cells are used as the donor nuclei.     

Efficient production of transgenic cloned calves using preimplantation screening

The genetic manipulation of donor cells before nuclear transfer (NT) enables prior selection for transgene integration. However, selection for genetically modified cells using antibiotic drugs often results in mixed populations, resulting in a mixture of transgenic and nontransgenic donor cells for NT. In this study, we attempted to develop efficient strategies for the generation of human bile salt-stimulated lipase (BSSL) transgenic cows. Preimplantation screening by either biopsy or green fluorescent protein (GFP) expression was used to detect NT-derived BSSL transgenic embryos to ensure that the calf born would be transgenic. We compared the development rates of NT-derived embryos from G418- and GFP-selected donor cells. There were no significant differences (P < 0.001) in cleavage rate (67.2% vs. 60.0%) and blastocyst formation rate (44.9% vs. 41.2%). We also compared the pregnancy rates of the G418/biopsy and GFP preimplantation screened NT-derived blastocysts. The Day 40 pregnancy rate of the G418/biopsy group (40%) was lower than that of the GFP group (57%), but the calf birth rate of the G418/biopsy group (40%) was higher than that of the GFP group (21%). Healthy BSSL transgenic calves were born after both screening processes. This is the first report of biopsy-screened cloned transgenic animals. The results suggest that both selection methods are useful for detecting transgenic NT embryos without negatively affecting their development into viable transgenic offspring.