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Background: The three-parent assisted reproductive technique may increase oocyte competence. Objective: In this case-control study, the suitability of germinal vesicle transfer (GVT), synchronous ooplasmic transfer (sOT), asynchronous ooplasmic transfer using cryopreserved MII oocyte (caOT), and asynchronous ooplasmic transfer using waste MII oocyte (waOT) for maturation of the human-aged non-surrounded nucleolus germinal vesicle-stage (NSN-GV) oocyte were investigated. Materials and Methods: NSN-GV oocytes were subjected to four methods: group A (GVT), B (sOT), C (caOT) D (waOT), and E (Control). The fusion rates, MI, MII, ICSI observations and cleavage at 2-cell, 4-cell, and 8-cell stages were compared in the groups. Results: In GVT, none of the oocytes fused. In sOT, all oocytes fused, 20 achieved the MI, 14 progressed to MII, 8 fertilized, 6 cleaved and 5, 4, and 3 achieved the 2- cells, 4-cells and 8-cells, respectively. In caOT, all oocytes fused and achieved the MI, 8 progressed to MII and fertilized, 6 cleaved and 6, 5, and 5 achieved the 2-cells, 4- cells, and 8-cells respectively. In waOT, all oocytes fused, 5 and 3 progressed to MI and MII, respectively, but only one fertilized, cleaved and reached a 4-cells stage. In group E, 6 and 2 oocytes progressed to MI and MII, respectively, and only one fertilized but arrested at the zygote stage. caOT had the highest survival rate when compared to sOT (p = 0.04), waOT (p = 0.002), and control (p = 0.001). Conclusion: The caOT method was beneficial over sOT, waOT, and GVT in supplementing the developmental capacity of human-aged NSN-GV oocytes. [ABSTRACT FROM AUTHOR]

Mitochondrial disease is a kind of genetic disease caused by the mutation in mitochondria genome. There is not a cure method for it. Recently, the nuclear transfer techniques including germinal vesicle transfer, spindle transfer, polar body transfer and pronuclear transfer provide a new hope for the treatment of mitochondrial disease. The germinal vesicle transfer requires the in vitro maturation of oocytes which may affect the development potential of oocytes. The spindle transfer may be at the high risk of damaging spindle since the spindle assembly is sensitive to mechanical stimulus and chromosomes are not enclosed within a nuclear membrane at this stage. As for the polar body transfer, the long-term side-effect is not clear since this technique steps over the natural selection. Pronuclear transfer, with the advantage of easy operation and sufficient research evidences, has the potential of clinical transformation in a short time，although there is still a ethical concern in the cytoplasmic donor from the zygote discarding itself genetic material. This paper reviews the possible application of four kinds of mitochondrial replacement techniques in the treatment of mitochondrial diseases. [ABSTRACT FROM AUTHOR]

Transgenic (TG) pigs are important in biomedical research and are used in disease modeling, pharmaceutical toxicity testing, and regenerative medicine. In this study, we constructed two vector systems by using the promoter of the pig glial fibrillary acidic protein (pGFAP) gene, which is an astrocyte cell marker. We established donor TG fibroblasts with pGFAP-CreERT2/LCMV-EGFPLoxP and evaluated the effect of the transgenes on TG-somatic cell nuclear transfer (SCNT) embryo development. Cleavage rates were not significantly different between control and transgene-donor groups. Embryo transfer was performed thrice just before ovulation of the surrogate sows. One sow delivered 5 TG piglets at 115 days after pregnancy. Polymerase chain reaction (PCR) analysis with genomic DNA isolated from skin tissues of TG pigs revealed that all 5 TG pigs had the transgenes. EGFP expression in all organs tested was confirmed by immunofluorescence staining and PCR. Real-time PCR analysis showed that pGFAP promoter-driven Cre fused to the mutated human ligand-binding domain of the estrogen receptor (CreERT2) mRNA was highly expressed in the cerebrum. Semi-nested PCR analysis revealed that CreERT2-mediated recombination was induced in cerebrum and cerebellum but not in skin. Thus, we successfully generated a TG pig with a 4-hydroxytamoxifen (TM)-inducible pGFAP-CreERT2/EGFPLoxP recombination system via SCNT. [ABSTRACT FROM AUTHOR]

Objective To investigate the expression profile of mRNAs in brain samples collected from pronuclear transfer (PNT) mice. Methods Female CD-1 mice were superovulated, and zygotes were collected after mating with adult male mice. Zygotes with two pronuclei were selected for pronuclear transfer manipulation, and then the reconstructed zygotes were transferred into the oviduct of pseudopregnant female mice. The infant mice obtained from pronuclear transfer were called PNT group, while the embryoes that were not performed pronuclear transfer was regarded as control group. Total RNA were extracted from brain samples of both PNT and control mice, and cDNA were labeled with fluorescent dye. Genes that were differentially expressed were identified using the Agilent mouse mRNA array. Gene ontology analysis and pathway analysis were also completed. Results Compared with control group, 392 mRNAs were expressed differentially, which showed more than 2.0 times variation and statistical significance, accounting for 1.7% of all mRNAs. Among those 366 mRNAs were up-regulated and 26 mRNAs were down-regulated. Eleven mRNAs came to 4.0 times variation in total. Gene ontology analysis indicated that differentially expressed genes were significantly enriched in alternative mRNA splicing, small GTPase mediated signal transduction, regulation of insulin receptor signaling pathway, hydrolase activity, transmembrane transporter activity and pyrophosphatase activity. Significant enriched pathway terms contained ion channel transport, fatty acid metabolism, butanoate metabolism, triacylglycerol and ketone body metabolism. Conclusion Pronuclear transfer might influence some key metabolism process in mouse brain. [ABSTRACT FROM AUTHOR]

A bstract The conversion of the nuclear program of a somatic cell from a differentiated to an undifferentiated state can be accomplished by transplanting its nucleus to an enucleated oocyte (somatic cell nuclear transfer [SCNT]) in a process termed 'reprogramming.' This process achieves pluripotency and occasionally also totipotency. Exploiting the obstacle of tetraploidy to full development in mammals, we show that mouse ooplasts transplanted with two somatic nuclei simultaneously (double SCNT) support preimplantation development and derivation of novel tetraploid SCNT embryonic stem cells (tNT-ESCs). Although the double SCNT embryos do not recapitulate the expression pattern of the pluripotency-associated gene Oct4 in fertilized embryos, derivative tNT-ESCs have characteristics of genuine pluripotency: in vitro they differentiate into neurons, cardiomyocytes, and endodermal cells; in vivo, tNT-ESCs form teratomas, albeit at reduced rates compared to diploid counterparts. Global transcriptome analysis revealed only few specific alterations, for example, in the quantitative expression of gastrulation-associated genes. In conclusion, we have shown that the oocyte's reprogramming capacity is in excess of a single nucleus and that double nucleus-transplanted embryos and derivative ESCs are very similar to their diploid counterparts. These results have key implications for reprogramming studies based on pluripotency: while reprogramming in the tetraploid state was known from fusion-mediated reprogramming and from fetal and adult hepatocyte-derived induced pluripotent stem cells, we have now accomplished it with enucleated oocytes. S tem C ells 2013;31:2343-2353 [ABSTRACT FROM AUTHOR]

Background: Over the past several years, mammals have been successfully cloned by either the splitting of an early stage embryo or nuclear transfer of adult somatic cells (NT) into oocytes. Although it has been 15 years since the generation of the first cloned mammals from somatic cells by NT, the success rate for producing live offspring by this technique is low regardless of the cell type and animal species used. However, these techniques have the potential to be important tools for future research in basic biology. In the present study, we described our experiences in producing successfully cloned mouse using NT method and piezo-actuated micromanipulator. Methods: B6D2F1 mice, 8-12 weeks old, were superovulated with injections of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin administered 48 hr apart. Enucleation and donor nuclei cumulus cell injection were performed with a piezo-actuated micromanipulator after which activation and trichostatin A treatment were used for reconstructed oocytes. Two-cell stage cloned embryos that developed in the mWM medium were transferred into the oviducts of pseudopregnant NMRI mice. Results: Of 367 oocytes collected, 131 (69%) developed into 2-cell stage embryos. Of these, 5 (1%) live pups were successfully delivered. We used NMRI foster mother to raise the pups by lactation. One adult cloned mouse was mated, after which she delivered and raised normal offspring. Conclusion: For mouse cloning, the present study also successfully tested the capability of somatic cell nuclear transfer SCNT using a piezo unit. [ABSTRACT FROM AUTHOR]

Abstract: Bone marrow stromal cells (BMSCs) are promising donor cells for transplantation therapies for a variety of diseases. However, there still lack efficient ways to induce directional differentiation of BMSCs to promote their practical use in transplantation therapy. In this study, we constructed hybrid cells by transferring neuronal nuclei into BMSC cytoplasts and investigated the proliferative capacity and phenotypic characteristics of the hybrid cells. The neuronal nuclei were labeled with Hoechst 33342 before the transfer process, and the cell membrane antigen CD71 was used as a marker of BMSC cytoplasts. The BMSC cytoplasts and neuronal karyoplasts were separated by Ficoll density gradient ultracentrifugation. The hybrid cells were generated by the polyethylene glycol-mediated fusion of BMSC cytoplasts with neuronal karyoplasts. The hybrid cells exhibited Hoechst 33342 staining in their nuclei and CD71 staining on their cytomembranes, which confirmed the success of cell fusion. The hybrid cells were positive for BrdU immunostaining. Viability analysis of the cultured hybrid cells by the MTT assay demonstrated their proliferative ability. Immunocytochemical staining revealed the expression of the neuron-specific markers NeuN and MAP2 in the third passage hybrid cells, which indicated their neuronal phenotypic characteristics. The results demonstrated that the hybrid cells produced by fusing neuronal karyoplasts with BMSC cytoplasts had proliferative capability and expressed the neuron-specific markers. Further study is required to investigate the phenotype of the hybrid cells both structurally and functionally. [Copyright &y& Elsevier]

Objective: To investigate the developmental potential of mouse somatic nuclear transfer embryos (SCNT) with different treatment of donor cell. Methods: Two methods, the intact cell and the naked nuclei injection, were used to produce cloned embryos. The reconstructed embryos and IVF control embryos were transferred into receptors' uterine cavity. The rates of activation, cleavage, blastocyst of reconstructed embryos and in vivo developmental outcomes were compared. Results: The rate of cleavage and blastocyst in the intact cell group was significantly lower than that in the bare nucleus group (χ²cleavage=5.785, P=0.016; χ²blastocyst 6=6.092, P=0.014). There were significantly difference in the weight of fetus in three groups F=14.331, P=0.00), the intact group had the heaviest wet weight of fetus. The weights of placental in the bare nuclear group and the IVF group were significantly lower than that in the intact cell group (compared with the normal group, t= -8.786, P=0.00; compared with bare nuclear group, t=-6.573, P=0.00). Conclusions: The developmental potential of mouse somatic cell nuclear transfer embryos could be improved by the bare nucleus injection. [ABSTRACT FROM AUTHOR]

Abstract: It is now over a decade since the birth, in 1996, of Dolly the first animal to be produced by nuclear transfer using an adult derived somatic cell as nuclear donor. Since this time similar techniques have been successfully applied to a range of species producing live offspring and allowing the development of transgenic technologies for agricultural, biotechnological and medical uses. However, though applicable to a range of species, overall, the efficiencies of development of healthy offspring remain low. The low frequency of successful development has been attributed to incomplete or inappropriate reprogramming of the transferred nuclear genome. Many studies have demonstrated that such reprogramming occurs by epigenetic mechanisms not involving alterations in DNA sequence, however, at present the molecular mechanisms underlying reprogramming are poorly defined. Since the birth of Dolly many studies have attempted to improve the frequency of development, this review will discuss the process of animal production by nuclear transfer and in particular changes in the methodology which have increased development and survival, simplified or increased robustness of the technique. Although much of the discussion is applicable across species, for simplicity we will concentrate primarily on published data for cattle, sheep, pigs and mice. [Copyright &y& Elsevier]