Blastocyst development after intergeneric nuclear transfer of mountain bongo antelope somatic cells into bovine oocytes

Intergeneric embryos were constructed by nuclear transfer using Mountain Bongo antelope somatic cells fused with enucleated bovine oocytes and their subsequent development in vitro was investigated. After two to six passages, starved or non-starved skin fibroblast cells were used as donor nuclei. In vitro matured bovine oocytes were enucleated by squeezing the first polar body and surrounding cytoplasm through a slit in the zona pellucida. After injection of a somatic cell into the perivitelline space, couplets were fused electrically and activated chemically, then subjected to different embryo culture treatments. Serum starvation had no effect on the frequency of cleavage to two cells or on development to the blastocyst stage in either sequential hamster embryo culture medium (HECM)-6/TCM-199 + serum or HECM-9/TC-199 + serum, or modified synthetic oviduct fluid (mSOF) culture medium. When couplets from non-starved donor nuclei were cultured, the frequency of cleavage (66 +/- 8% vs. 44 +/- 5%), development to >/=9 cells (46 +/- 6% vs. 24 +/- 4%), and formation of blastocysts (24 +/- 5% vs. 11 +/- 2%) were all significantly higher (p < 0.05) in the HECM-6 medium than in mSOF medium. In conclusion, bovine oocytes can support blastocyst development after intergeneric fusion with bongo fibroblasts. This technique could potentially be used as an alternative to using scarce bongo oocytes in attempts to propagate these endangered animals.     

Freeze-dried somatic cells direct embryonic development after nuclear transfer

The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies.     

绵羊体细胞核移植去核前程序的优化

目前绵羊体细胞克隆效率仍然很低,本研究拟对去核前的操作环节进行优化。主要为卵巢保存时间(3 h和3–5 h)、卵母细胞体外成熟时间(18 h和24 h)、供核细胞贴壁率(10%和30%)和盲吸法去核时间(16 hpm和18 hpm)等4个方面优化。以成熟率、融合率和重构胚胎发育能力作为评价参数。结果表明:在卵巢保存方面,卵巢保存3 h组卵母细胞成熟率显著高于3–5 h组卵母细胞成熟率(60.18%vs 52.50%)(P0.05),重构胚胎发育力差异不显著(P0.05);在体外成熟时间方面,体外成熟18 h组和24 h组卵母细胞成熟率差异极显著(53.81%vs 89.06%)(P0.01),胚胎发育力差异不显著(P0.05);在融合率方面,贴壁率30%组极显著高于贴壁率10%组(80.85%vs 57.69%)(P0.01),在克隆胚胎发育率方面没有显著差异(P0.05),具有贴比率差异性的细胞在细胞生长平台期表现出差异性;在去核时间方面,16 hpm组和18 hpm组胚胎卵裂率差异显著,囊胚发育力差异不显著(P0.05),16 hpm组获得一只克隆羊,重复16 hpm获得4只妊娠克隆羊。组织微卫星序列经SDS-PAGE分析,DNA指纹与供体细胞相同。结论:去核前程序的优化保证了材料的质量,为提高克隆胚胎数量和质量奠定基础,可以获得体细胞克隆羊。     

Production of cloned goats after nuclear transfer using adult somatic cells.

The developmental potential of adult somatic nuclei after nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated in a dwarf breed of goat (BELE: Breed Early Lactate Early). Somatic donor cells were obtained from two different sources: 1) adult granulosa cells (GCs) and 2) fetal fibroblasts. Primary GCs were obtained from follicular aspirants after laparoscopic oocyte pick-up (LOPU) and were cryopreserved immediately. Frozen aliquots of cells were thawed and cultured until confluent and were then cultured in low serum for 4 days before use in NT. Immature oocytes were obtained by LOPU and matured before enucleation and NT. Ninety-one adult GC-derived NT embryos were transferred into eight recipients, four of which were confirmed pregnant (50%) at Day 30 by ultrasound. Fifty-four male fetal fibroblast-derived NT embryos were transferred into six recipients, one of which was confirmed pregnant (17%). All pregnancies were maintained through term. Four recipients delivered seven female kids (three sets of twins) derived from the GC cultures (7.7% of embryos transferred). The other recipient delivered two male kids (3.7% of embryos transferred). Birth weights were within the normal range for dwarf goats. One female twin and one male twin died at birth; the remaining kids appeared healthy and normal. DNA analysis confirmed that the kids were genetically identical to their respective donors. These results demonstrated that adult caprine somatic cells could direct normal development after NT.     

体细胞核移植后核重编程的影响因素

近年来,人类核移植胚胎干细胞建系成为一项炙手可热的研究,用再生医学的理念治疗退行性疾病及器官移植为这一研究带来无穷的魅力和生命力;但是核重编程仍是核移植技术的瓶颈,制约了重构胚胎干细胞的研究。核重编程是指供体细胞核移入卵母细胞后必须停止本身的基因表达程序并恢复为胚胎发育所必需的特定的胚胎表达程序。只有供核发生完全重编程,重构胚胎才能正常发育。核重编程与供核者的年龄,供核细胞的组织来源、分化状态、细胞周期、传代次数,供核的表遗传标记以及供卵者的年龄、卵子的成熟度等因素有关。一般来说,颗粒细胞作为核供体最易被核重编程。供核者为胎体或新生体,供核细胞处于低分化状态或已传数代,供核细胞经过去表遗传标记处理,供卵者性成熟且年龄轻、卵子核与胞浆都成熟等均为有利于核重编程的因素。重构胚胎的培养方法对核重编程也至关重要,目前主张使用序贯培养及体细胞化培养。创造各种适于核重编程的条件有利于从更高的起点开展核移植胚胎干细胞研究,提高重构胚胎干细胞建系效率。     

Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes

To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and fi0 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of thedonor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PCR and immunocytochemistry with probes that distinguish between the various species. The ntES ceils maintainthe capability of sustained growth in an undifferen tiated state, and form embryoid bodies, which, on furtherinduction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that expressmarkers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NTto rabbit eggs retain phenotypes similar to those of conventional human ES ceils, including the ability toundergo multilineage cellular differentiation.     

Evaluation of gene targeting by homologous recombination in ovine somatic cells

Mouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post-natal fibroblasts generated G418-resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post-natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability.     

Nuclear transfer-mediated rescue of the nuclear genome of nonviable mouse cells frozen without cryoprotectant

Nuclear transfer (NT) provides an opportunity for clonal amplification of a nuclear genome of interest. Here, we report NT-mediated reprogramming with frozen mouse cells that were nonviable because they were frozen at -80 degrees C for up to 342 days without a cryoprotectant. We derived eight embryonic stem (ES) cell lines from cloned blastocysts by conventional NT procedure and five ntES (nuclear transfer embryonic stem) cell lines by a modified NT procedure in which a whole cell instead of a nucleus was injected into an enucleated oocyte. Chromosome analysis revealed that 12 of 13 ntES cell lines have normal karyotypes. On injection of ntES cells into tetraploid blastocysts to generate clonal mice that are nearly completely ntES-cell derived, live pups were obtained; four clonal mice survived until adulthood. On injection of ntES cells into diploid blastocysts, chimeric mice with a high somatic ES cell contribution were generated; germ-line transmission was obtained. Our findings indicate that chromosome stability and genomic integrity can be maintained in mouse somatic cells after freezing without cryoprotection and that NT and ES cell techniques can rescue the genome of these cells.     

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

Background  

The mitochondrial DNA (mtDNA) of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT) was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts.     

牛体细胞核移植显微操作环节的优化

本研究从牛卵母细胞去核方法(纺锤体观测仪法&Hoechst33342染色法)、供体细胞核引入去核卵细胞质的方法(卵细胞质注射法和电融合法)和重构胚胎电融合(3组参数)等3个环节对牛体细胞核移植的显微操作过程及相关参数进行了筛选优化。以核移植胚胎的卵裂率、囊胚发育率作为检测指标,对不同的方法所获得的克隆胚胎的卵分裂率与囊胚发育率进行比较,最后筛选获得1个优化的牛体细胞核移植操作程序,即采用Spindle view系统对牛卵母细胞进行去核操作,将供核体细胞注射到卵周隙,然后通过电融合法将供体核引入去核卵细胞质(电融合参数为1.9kV/cm,脉冲时程10μs,方波2次间隔2s)。以此核移植程序进行牛体细胞核移植实验,自获得克隆胚胎中筛选80枚优质囊胚移植到33头受体牛子宫内,最后2头母牛产下2头克隆牛犊,结果表明利用该优化的显微操作环节进行牛体细胞核移植可以获得体细胞克隆牛犊。     

体细胞重编程为多能干细胞的研究进展

体细胞通过重编程转变成其他类型的细胞,在再生医学方面具有重要的应用前景。细胞重编程的方法主要有体细胞核移植、细胞融合、细胞提取物诱导、限定因子诱导等,这些方法可以不同程度地改变细胞命运。最近,限定因子诱导的多能干细胞（induced pluripotent stem cell。iPS）为重编程提供了一种崭新的方法,不仅可以避免伦理争议,还提供了一种更为便利的技术,为再生医学开辟了新的天地;同时,iPS技术为研究基因表达调控、蛋白质互作、机体生长发育等提供了一个非常重要的研究手段。本文主要论述了体细胞重编程的方法及iPS细胞的进展、面临的问题和应用前景。     

Normal specification of the extraembryonic lineage after somatic nuclear transfer

To examine the establishment and maintenance of trophectoderm (TE) lineage in somatic cloned blastocysts, the expression of Cdx2, a key molecule for specification of TE fate, was immunohistochemically examined simultaneously with Oct4 expression. Cloned mouse embryos were made by nuclear transfer using cumulus cells, tail-tip fibroblasts, and embryonic stem cells. After 96 h of culture, the rates of Oct4-expressing blastocysts were as low as 50% and 60% for cumulus and fibroblast clones, respectively. However, regardless of Oct4 expression, the majority of those cloned blastocysts (> 90%) normally expressed Cdx2. Thus, even though somatic cloned embryos have reduced potential to produce the inner cell mass lineage, the TE lineage can be established and maintained.     

Organisation of complex nuclear domains in somatic mouse cells.

The number and associations of heterochromatin chromocenters, nucleoli, centromeres and telomeres were studied in the nucleus of different somatic cells of Mus domesticus. Fibroblasts of the cell line 3T3, kidney cells (primary culture), and bone marrow cells were used. The above mentioned nuclear and chromosome markers were identified by DAPI/actinomycin D, indirect immunofluorescence with anti-centromere antibodies, silver impregnation for nucleolar proteins and fluorescence in situ hybridisation (FISH) with telomeric probes. The quantitative analysis of the nuclei showed that the pericentromeric heterochromatin is organised in about 18 chromocenters per nucleus in the 3T3 cells, and about seven in kidney and bone marrow cells, having generally a peripheral distribution in the nucleus of all the studied cells. Several aggregated centromeres were participating in each of the chromocenters, about four centromeres per 3T3 cell and about six centromeres per kidney and bone marrow cells. Some of the chromocenters were also in close association with nucleoli. The number of telomeric labels per nucleus was as expected for each chromosome set (2n = 68-70 and 2n = 40). About half of the telomeric signals were loosely aggregated within the heterochromatic blocks while the rest were distributed in the nucleus as unrelated units not bound with chromocenters. The three cell types have complex nuclear territories formed by different chromosomal domains: the pericentromeric heterochromatin, centromeres, proximal telomeres and nucleoli. With the exception of some bone marrow cells, we have not found a nuclear polarisation of the analysed chromosomal markers compatible with the Rabl configuration. However, Rabl anaphasic polarisation allows the contact of centromeric regions making possible that centromeric associations arise. If in addition, associative elements such as constitutive heterochromatin or nucleoli are close to the centromeric regions, like in Mus domesticus chromosomes, then the associations might be consolidated and persist until the interphase. These associations may be the origin of the nuclear domains described here for Mus domesticus somatic cells.     

Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos

Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (P < 0.05). The use of donor cells of any type in later passages decreased the rate of blastocyst formation. Treatment with trichostatin-A did not improve the rate of blastocyst formation from cleaved dewclaw cell-derived embryos but did so in the embryos derived from the tail-tip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.     

Centrifugal separation of irradiated cultures of Escherichia coli cells into viable and nonviable populations.

Incubation of ultraviolet-irradiated Escherichia coli B/r cultures with 0.7% Triton X-100 resulted in a large decrease in turbidity. Under phase-contrast optics, most of the irradiated detergent-treated cells were smaller than normal and of low phase density; only a small percentage were normal or larger than normal and of normal phase density. Irradiated cells not treated with detergent showed fewer pronounced morphological changes. Irradiated cells treated with detergent lost large amounts of proteins and ribonucleic acid, but not of deoxyribonucleic acid. Such cultures could be separated by centrifugation into populations of (i) slowly sedimenting cells consisting of small, phase-light cells of low viability and (ii) large cells of normal phase density and high viability (100%). A similar separation was effected in gamma-irradiated cultures.     

Lyophilized somatic cells direct embryonic development after whole cell intracytoplasmic injection into pig oocytes

The study investigated the feasibility of lyophilization for long-term preservation of somatic cells and embryonic development after whole cell intracytoplasmic injection (WCICI) into enucleated pig oocyte. Confluent cultured porcine fetal fibroblast (pFF) cells were lyophilized and stored at 4 °C for at least 6 months. Results showed that compared to non-lyophilized control cells, lyophilized cells had drastically reduced cellular viability (P < 0.01). WCICI of reconstituted lyophilized cells could support complete embryonic development. However, the rates of cleavage (64.7 ± 2.7 vs. 43.5 ± 4.7%) and blastocyst formation (18.2 ± 0.6 vs. 10.2 ± 1.6%) were lower than that of control (P < 0.05). Total nuclei number per blastocyst (30.4 ± 4.5 vs. 25.2 ± 4.7) and intensity of acetylation at histone H3 (AcH3) protein (55.9 ± 3.5 vs. 53.3 ± 3.8) did not differ (P > 0.05). The development ability of embryos, produced from lyophilized somatic cells, was further increased (19.5 ± 2.4 vs. 10.2 ± 1.6%; P < 0.05) by treatment with trichostatin A (TSA) for 24 h post-activation. These TSA-treated embryos also had AcH3 level comparable with in vitro fertilized embryos (63.1 ± 3.2 vs. 69.9 ± 1.3). In conclusion, our results suggest that lyophilized somatic cells can direct embryonic development up to blastocyst stage after WCICI into pig oocytes. Treatment of embryos, produced from lyophilized somatic cells, with TSA can further increase their in vitro developmental potential.     

Gene transfer into zebrafish by sperm nuclear transplantation

A technique for fertilizing zebrafish eggs by injection of sperm nuclei is described. Eggs that cleave normally can develop into swimming larvae and give rise to fertile adults. If sperm nuclei are preincubated for 20 min with DNA encoding the green fluorescent protein, transgene expression can be detected in all cells of the embryo. The use of condensed sperm nuclei allows injection with a small bore pipette, which is critical for successful injection of the relatively small zebrafish egg. This technique enables the generation of ubiquitously expressing transgenic zebrafish directly by microinjection. Hence, experiments involving transgenic fish can be completed in days, without the need for growing and breeding founders. This technique may also be used to generate transgenic lines, as transgene expression was visible in the offspring of transgenic founders. The method described here is likely to be applicable to other teleosts, such as medaka and salmon.     

Cloned piglets born after nuclear transplantation of embryonic blastomeres into porcine oocytes matured in vivo

Nuclear transplantation in the pig is more difficult than in other domestic animals and only one embryonic nuclear transplantation (NT) pig has been born to date. In this study, reconstituted porcine embryos were produced by electrofusion of blastomeres from in vivo four-cell embryos to enucleated in vivo or in vitro matured (IVM) oocytes. Nuclear transfer using cumulus cells as nuclear donors was also conducted. When blastomeres were used as donors, the electrofusion rate was significantly higher in oocytes matured in vivo (91.5%) than in those matured in vitro (66.1%) (p < 0.01). After fusion, the NT embryos reconstituted from in vivo matured oocytes developed to blastocysts at a rate of 10.3% after culture in rabbit oviducts for up to 5 days, while only 5.9% of the NT embryos reconstructed from in vitro matured oocytes developed to blastocyst stage. Electrofusion rate of cumulus cell nuclei with enucleated IVM oocytes was lower (47.6%) and only 1.5% (2/136) of the reconstituted eggs developed in vitro to morula stage, and 1.9% developed to blastocysts when cultured in the ligated rabbit oviducts. Transfer of 94 embryos reconstructed by blastomere NT with in vivo matured oocytes to five synchronous recipients resulted in the birth of two cloned piglets. No piglet was born following transfer to two recipients of embryos (n = 39) derived from NT with in vitro matured oocytes. The results demonstrate that in vivo matured oocytes are better recipients than those matured in vitro for pig cloning.     

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%).