Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos.

We investigated the influence of the cell cycle stage of the nuclear donor on prematurely condensed chromatin (PCC) and spindle morphology and on chromosome constitution in rabbit nuclear transplant embryos. The configuration of PCC following nuclear transplantation with G1, early S, and late S phase donor nuclei (G1, early S, and late S transplants, respectively) was characterized in whole mounts and chromosome spreads. In addition, the influence of the donor cell cycle stage on chromosome constitution in cleavage stage-manipulated embryos was determined. Within 2 h after fusion of the donor blastomere, the recipient oocyte cytoplasm was able to induce formation de novo of a metaphase plate associated with a spindle in G1, early S, and late S transplants. Metaphase chromosomes and spindle were intact in most cases of PCC in G1 transplants. However, these structures displayed minor abnormalities in early S transplants and gross abnormalities in late S transplants, such as incomplete or absent spindle formation and incomplete chromatin condensation. Normal chromosomes were present in G1 and early S transplants, whereas chromosome abnormalities were detected in late S transplants. The results indicate that morphology of prematurely condensed G1 and early S chromatin has a minor influence on chromosome constitution of manipulated embryos. That of late S chromatin, however, affects chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late S phase donor nuclei are used.     

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.     

人-兔异种核移植构建克隆胚的实验研究

“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著（P〈0．05）;不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人一兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。     

Cloning to reproduce desired genotypes

Cloned sheep, cattle, goats, pigs and mice have now been produced using somatic cells for nuclear transplantation. Animal cloning is still very inefficient with on average less than 10% of the cloned embryos transferred resulting in a live offspring. However successful cloning of a variety of different species and by a number of different laboratory groups has generated tremendous interest in reproducing desired genotypes. Some of these specific genotypes represent animal cell lines that have been genetically modified. In other cases there is a significant demand for cloning animals characterized by their inherent genetic value, for example prize livestock, household pets and rare or endangered species. A number of different variables may influence the ability to reproduce a specific genotype by cloning. These include species, source of recipient ova, cell type of nuclei donor, treatment of donor cells prior to nuclear transfer, and the techniques employed for nuclear transfer. At present, there is no solid evidence that suggests cloning will be limited to only a few specific animals, and in fact, most data collected to date suggests cloning will be applicable to a wide variety of different animals. The ability to reproduce any desired genotype by cloning will ultimately depend on the amount of time and resources invested in research.     

Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells

To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.     

Developmental ability of bovine embryos after nuclear transfer based on the nuclear source: in vivo versus in vitro

Bovine nuclear transfer embryos reconsitituted from in vitro-matured recipient oocyte cytoplasm and different sources of donor nuclei (in vivo, in vitro-produced or frozen-thawed) were evaluated for their ability to develop in vitro. Their cleavage rate and blastocyst formation are compared with those of control IVF embryos derived from the same batches of in vitro-matured oocytes that were used for nuclear transfer and were co-cultured under the same conditions on bovine oviducal epithelial cell monolayers for 7 d. Using fresh donor morulae as the source of nuclei resulted in 30.2% blastocyst formation (150 497 ), which was similar to that of control IVM-IVF embryos (33.8% blastocysts, 222 657 ). When IVF embryos were used as the source of nuclei for cloning, a slightly lower blastocyst formation rate (22.6%, 41 181 ) was obtained but not significantly different from that using fresh donor morulae. Nuclear transfer embryos derived from vitrified donor embryos showed poor development in vitro (7.1%, 11 154 ). No difference in morphology or cell number was observed after 7 d of co-culture between blastocysts derived from nuclear transfer or control IVF embryos. The viability of 34 in vitro-developed nuclear transfer blastocysts was tested in vivo and resulted in the birth of 11 live calves (32.3%).     

Improvements in cloning efficiencies may be possible by increasing uniformity in recipient oocytes and donor cells

The low efficiency of somatic cell cloning is the major obstacle to widespread use of this technology. Incomplete nuclear reprogramming following the transfer of donor nuclei into recipient oocytes has been implicated as a primary reason for the low efficiency of the cloning procedure. The mechanisms and factors that affect the progression of the nuclear reprogramming process have not been completely elucidated, but the identification of these factors and their subsequent manipulation would increase cloning efficiency. At present, many groups are studying donor nucleus reprogramming. Here, we present an approach in which the efficiency of producing viable offspring is improved by selecting recipient oocytes and donor cells that will produce cloned embryos with functionally reprogrammed nuclei. This approach will produce information useful in future studies aimed at further deciphering the nuclear reprogramming process.     

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P＜0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.     

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.     

Production of a cloned calf using zona-free serial nuclear transfer

The efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos. This procedure involves a second nuclear transfer step; pronuclear-like cloned nuclei are transferred into pronuclear stage zygotic cytoplasts. The present study reports on the development of a serial nuclear transfer technique in the bovine, based on a zona-free method (hand-made cloning), resulting in the birth of a cloned calf. Comparisons were made between embryos produced by hand-made cloning and serial nuclear transfer. There were no differences between in vitro development or differential cell counts in the blastocysts produced. Transfer of 16 serial hand-made cloned blastocysts resulted in the production of one healthy calf (6%), whereas hand-made cloning resulted in the birth of 1 calf from 23 transferred blastocysts (4%). One serial nuclear transfer pre-term fetus had renal and hepatic abnormalities (previously observed in clones from this cell line). Although it may not be as beneficial in the bovine as in other species, normal placentation (size, placentomes and umbilicus) was encouraging. Refinement of this technique may help to identify species-specific differences in zygotic competence that affect reprogramming of donor cell nuclei and that may improve efficiency.     

Birth of mice after nuclear transfer by electrofusion using tail tip cells

Mice have been successfully cloned from cumulus cells, fibroblast cells, embryonic stem cells, and immature Sertoli cells only after direct injection of their nuclei into enucleated oocytes. This technical feature of mouse nuclear transfer differentiates it from that used in domestic species, where electrofusion is routinely used for nuclear transfer. To examine whether nuclear transfer by electrofusion can be applied to somatic cell cloning in the mouse, we electrofused tail tip fibroblast cells with enucleated oocytes, and then assessed the subsequent in vitro and in vivo development of the reconstructed embryos. The rate of successful nuclear transfer (fusion and nuclear formation) was 68.8% (753/1094) and the rate of development into morulae/blastocysts was 40.8% (260/637). After embryo transfer, seven (six males and one female; 2.5% per transfer) normal fetuses were obtained at 17.5-21.5 dpc. These rates of development in vitro and in vivo are not significantly different from those after cloning by injection (44.7% to morulae/blastocysts and 4.8% to term). These results indicate that nuclear transfer by electrofusion is practical for mouse somatic cell cloning and provide an alternative method when injection of donor nuclei into recipient oocytes is technically difficult.     

Telophase-stage host ooplasts support complete reprogramming of roscovitine-treated somatic cell nuclei in cattle

Nuclear-cytoplasmic incompatibilities are known to play a significant role in the developmental outcome of embryos produced by nuclear transfer, particularly when metaphase arrested oocytes are used as hosts for interphase donor nuclei. To further our understanding of how cell cycle coordination affects somatic cell cloning, somatic cells at different stages of the cell cycle were fused to host oocytes either before (metaphase II, M-II) or after (telophase II, T-II) activation. To obtain cells at different stages of the cell cycle, fetal fibroblast (FF) and granulosa cells (GC) were treated with roscovitine, an inhibitor of cyclin-dependent kinases (CDKs) resulting in a large percentage of cells in S/G(2)-phase. In contrast to the M-II group, which did better with confluent cells, embryos reconstructed with T-II cytoplasts resulted in higher rates of blastocyst formation when fused to cells recovered at 16-24 h after passage. Embryos reconstructed with FF treated with roscovitine and T-II cytoplasts (Rosc/T-II) resulted in similar blastocyst rate compared to those produced with confluent cells and M-II cytoplasts (Conf/M-II). Transfer of blastocysts to surrogate heifers resulted pregnancies and birth of healthy calves from Rosc/T-II and Conf/M-II reconstructed embryos. These results indicate that, when combined with nuclear donor cells at specific cell cycle stages, M-II and T-II bovine oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei.     

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.     

Global gene expression analysis of bovine blastocysts produced by multiple methods

Reproductive efficiency using somatic cell nuclear transfer (SCNT) technology remains suboptimal. Of the various efforts to improve the efficiency, chromatin transfer (CT) and clone-clone aggregation (NTagg) have been reported to produce live cloned animals. To better understand the molecular mechanisms of somatic cell reprogramming during SCNT and assess the various SCNT methods on the molecular level, we performed gene expression analysis on bovine blastocysts produced via standard nuclear transfer (NT), CT, NTagg, in vitro fertilization (IVF), and artificial insemination (AI), as well as on somatic donor cells, using bovine genome arrays. The expression profiles of SCNT (NT, CT, NTagg) embryos were compared with IVF and AI embryos as well as donor cells. NT and CT embryos have indistinguishable gene expression patterns. In comparison to IVF or AI embryos, the number of differentially expressed genes in NTagg embryos is significantly higher than in NT and CT embryos. Genes that were differentially expressed between all the SCNT embryos and IVF or AI embryos are identified. Compared to AI embryos, more than half of the genes found deregulated between SCNT and AI embryos appear to be the result of in vitro culture alone. The results indicate that although SCNT methods have altered differentiated somatic nuclei gene expression to more closely resemble that of embryonic nuclei, combination of insufficient reprogramming and in vitro culture condition compromise the developmental potential of SCNT embryos. This is the first set of comprehensive data for analyzing the molecular impact of various nuclear transfer methods on bovine pre-implantation embryos.     

Comparison of microinjection (piezo-electric) and cell fusion for nuclear transfer success with different cell types in cattle

Amongst the many variables that can determine success of cloning, the source of nuclei, the procedure used for nuclear transfer, and the activation of the reconstructed embryo are very important aspects. In this study, we have compared the two most common procedures for transferring nuclei to enucleated oocytes--cell fusion (CF) and piezoelectric microinjection (PEM) using different somatic cells--and we have investigated the effect of different activation procedures. Granulosa cells and fibroblasts were grown to confluency or in low serum to induce a quiescent state, while lymphocytes were thawed immediately prior to use. Enucleated oocytes were reconstructed either with CF or PME by 21-23 h postmaturation. For cell fusion, one pulse of 1 kVolt/cm for 30 microsec was used; for PEM, the cell membrane was broken by repeated pipetting and transferred in a 12% PVP solution to facilitate injection. Manipulated oocytes were activated with ionomycin and cycloheximide (CHX) or 6-DMAP (DMAP) and cultured in microdrops of SOF-BSA-AA. On day 7 (day 0: nuclear transfer), embryo development was evaluated and embryos were either transferred fresh or were frozen. More embryos were successfully reconstructed with PEM than CF, but a higher number of reconstructed embryos by CF developed to blastocyst at D + 7. In addition, in both systems more embryos were obtained after activation with DMAP than with CHX. The transfer of 141 embryos to recipients resulted in a pregnancy rate of 50%, and no differences were observed between the source of donor cell, the reconstruction methods, or the activation protocol. Six calves were delivered at term, and four survived. High pregnancy losses were observed throughout the gestation period.     

Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos

During differentiation, somatic nuclei acquire highly specialized DNA and chromatin modifications, which are thought to result in cellular memory of the differentiated state. Upon somatic nuclear transfer into oocytes, the donor nucleus may have to undergo reprogramming of these epigenetic marks in order to achieve totipotency. This may involve changes in epigenetic features similar to those that occur in normal embryos during early development. However, there is accumulating evidence that epigenetic reprogramming is severely deficient in cloned embryos. Several reports reveal inefficient demethylation and inappropriate reestablishment of DNA methylation in quantitative and qualitative patterns on somatic nuclear transfer. Here we examine histone H3 lysine 9 (H3-K9) methylation and acetylation in normal embryos and in those created by somatic nuclear transfer. We find that H3-K9 methylation is reprogrammed in parallel with DNA methylation in normal embryos. However, the majority of cloned embryos exhibit H3-K9 hypermethylation associated with DNA hypermethylation, suggesting a genome-wide failure of reprogramming. Strikingly, the precise epigenotype in cloned embryos depends on the donor cell type, and the proportion of embryos with normal epigenotypes correlates closely with the proportion developing to the blastocyst stage. These results suggest a mechanistic link between DNA and histone methylation in the mammalian embryo and reveal an association between epigenetic marks and developmental potential of cloned embryos.     

The possible role of cell cycle stage in mammalian cloning

Progress in mammalian cloning started from cloning embryos (of mice, rats, rabbits, sheep, goats, pigs, cattle and rhesus monkeys) and culminated in obtaining clones of sheep, cattle, pigs and mice from adult somatic cells. Knowing the relationship between the cell cycles of the recipient and the donor of cell nucleus in embryonic cloning by nuclear transfer one can adjust the phases of the cell cycle properly. Metaphase II recipients accept G1 (in most species) or G2 donors (in the mouse). Interphase recipients can harbour nuclei in all stages of cell cycle. Relatively little is known about somatic cloning. Two attitudes are applied: either the donor is in the G0 phase or the recipient is in a prolonged MII phase.