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Conservation of avian genetic resources is important, as 13.0% (1,313/10,064) of known avian species are currently categorized as endangered by the International Union for Conservation of Nature and Natural Resources (IUCN, 2012). Development of an integrated system for conservation of avian genetics is necessary; however, germplasm conservation systems developed for mammals cannot be applied directly to avians, primarily due to the unique anatomical and physiological characteristics of the avian egg. Thus an alternative strategy for conservation of oviparous species of animals must be developed. Recent technological developments for producing germline chimeras by the transfer of primordial germ cells into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. Furthermore, the possibility of propagating endangered wild birds through interspecies germline chimeras has been proposed. In the present review, the current status of avian genetic resource conservation is presented with a focus on the following 4 topics: 1) semen cryopreservation, 2) germline chimeras, 3) somatic nuclear transfer, and 4) other related technologies. [ABSTRACT FROM AUTHOR]

The article focuses on experiments which demonstrate cloning of piglets from induced pluripotent stem cells (iPSCs). It is mentioned that two cell lines were induced from porcine bone marrow cells and porcine ear fibroblasts with a use of lentiviral vector. The experiments show that iPSCs from pigs can be used for nuclear transfer as donors by silencing of exogenous transcription factors by various methods.

Contents Somatic cell nuclear transfer (SCNT) is now an established procedure used in cloning of several species. SCNT in dogs involves multiple steps including the removal of the nuclear material, injection of a donor cell, fusion, activation of the reconstructed oocytes and finally transfer to a synchronized female recipient. There are therefore many factors that contribute to cloning efficiency. By performing a retrospective analysis of 2005-2012 published papers regarding dog cloning, we define the optimum procedure and summarize the specific feature for dog cloning. [ABSTRACT FROM AUTHOR]

Abstract: The objective was to establish an efficient defined culture medium for bovine somatic cell nuclear transfer (SCNT) embryos. In this study, modified synthetic oviductal fluid (mSOF) without bovine serum albumin (BSA) was used as the basic culture medium (BCM), whereas the control medium was BCM with BSA. In Experiment 1, adding polyvinyl alcohol (PVA) to BCM supported development of SCNT embryos to blastocyst stage, but blastocyst formation rate and blastocyst cell number were both lower (P < 0.05) compared to the undefined group (6.1 vs. 32.6% and 67.3 ± 3.4 vs. 109.3 ± 4.5, respectively). In Experiment 2, myo-inositol, a combination of insulin, transferrin and selenium (ITS), and epidermal growth factor (EGF) were added separately to PVA-supplemented BCM. The blastocyst formation rate and blastocyst cell number of those three groups were dramatically improved compared with that of PVA-supplemented group in Experiment 1 (18.5, 23.0, 24.1 vs. 6.1% and 82.7 ± 2.0, 84.3 ± 4.2, 95.3 ± 3.8 vs. 67.3 ± 3.4, respectively, P < 0.05), but were still lower compared with that of undefined group (33.7% and 113.8 ± 3.4, P < 0.05). In Experiment 3, when a combination of myo-inositol, ITS and EGF were added to PVA-supplemented BCM, blastocyst formation rate and blastocyst cell number were similar to that of undefined group (30.4 vs. 31.1% and 109.3 ± 4.4 vs. 112.0 ± 3.6, P > 0.05). In Experiment 4, when blastocysts were cryopreserved and subsequently thawed, there were no significant differences between the optimized defined group (Experiment 3) and undefined group in survival rate and 24 and 48 h hatching blastocyst rates. Furthermore, there were no significant differences in expression levels of H19, HSP70 and BAX in blastocysts derived from optimized defined medium and undefined medium, although the relative expression abundance of IGF-2 was significantly decreased in the former. In conclusion, a defined culture medium containing PVA, myo-inositol, ITS, and EGF supported in vitro development of bovine SCNT embryos. [Copyright &y& Elsevier]

Cell size varies widely among different organisms as well as within the same organism in different tissue types and during development, which places variable metabolic and functional demands on organelles and internal structures. A fundamental question is how essential subcellular components scale to accommodate cell size differences. Nuclear transport has emerged as a conserved means of scaling nuclear size. A meiotic spindle scaling factor has been identified as the microtubule-severing protein katanin, which is differentially regulated by phosphorylation in two different-sized frog species. Anaphase mechanisms and levels of chromatin compaction both act to coordinate cell size with spindle and chromosome dimensions to ensure accurate genome distribution during cell division. Scaling relationships and mechanisms for many membrane-bound compartments remain largely unknown and are complicated by their heterogeneity and dynamic nature. This review summarizes cell and organelle size relationships and the experimental approaches that have elucidated mechanisms of intracellular scaling. [ABSTRACT FROM AUTHOR]

Pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-1 β and TNF-α predominate in inflammatory bowel diseases (IBD) and TNBS colitis. Increased levels of serine proteases activating protease-activated receptor 2 (PAR-2) are found in the lumen and colonic tissue of IBD patients. PAR-2 activity and pro-inflammatory cytokines impair epithelial barrier, facilitating the uptake of luminal aggressors that perpetuate inflammation and visceral pain. Soy extracts contain phytoestrogens (isoflavones) and serine protease inhibitors namely Bowman-Birk Inhibitors (BBI). Since estrogens exhibit anti-inflammatory and epithelial barrier enhancing properties, and that a BBI concentrate improves ulcerative colitis, we aimed to evaluate if a fermented soy germ extract (FSG) with standardized isoflavone profile and stable BBI content exert cumulative or synergistic protection based on protease inhibition and estrogen receptor (ER)-ligand activity in colitic rats. Female rats received orally for 15 d either vehicle or FSG with or without an ER antagonist ICI 182.780 before TNBS intracolonic instillation. Macroscopic and microscopic damages, myeloperoxidase activity, cytokine levels, intestinal paracellular permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression were assessed 24 h, 3 d and 5 d post-TNBS. FSG treatment improved the severity of colitis, by decreasing the TNBS-induced rise in gut permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression at all post-TNBS points. All FSG effects were reversed by the ICI 182.780 except the decrease in faecal proteolytic activity and PAR-2 expression. In conclusion, the antiinflammatory properties of FSG treatment result from two distinct but synergic pathways i.e an ER-ligand and a PAR-2 mediated pathway, providing rationale for potential use as adjuvant therapy in IBD. [ABSTRACT FROM AUTHOR]

Early interspecies nuclear transfer (iNT) experiments suggested that a foreign nucleus may become permanently damaged after a few rounds of cell division in the cytoplasm of another species. That is, in some distant species combinations, nucleocytoplasmic hybrid (cybrid) blastula nuclei can no longer support development, even if they are back-transferred into their own kind of egg cytoplasm. We monitored foreign DNA amplification and RNA production by quantitative PCR (qPCR) and RT-qPCR in interorder amphibian hybrids and cybrids formed by the transfer of newt (Pleurodeles waltl) embryonic nuclei into intact and enucleated frog (Xenopus laevis) eggs. We found a dramatic reduction in the expansion of foreign DNA and cell numbers in developing cybrid embryos that correlated with reduced gene transcription. Interestingly, expansion in cell numbers was rescued by the recipient species (Xenopus) maternal genome in iNT hybrids, but it did not improve R waltl DNA expansion or gene transcription. Also, foreign gene transcripts, normalized to DNA copy numbers, were mostly normal in both iNT hybrids and cybrids. Thus, incomplete foreign DNA replication and/or chromosome segregation during cell division may be the major form of nuclear damage occurring as a result of nuclear replication in a foreign cytoplasmic environment. It also shows that the mechanisms of embryonic gene transcription are highly conserved across amphibians and may not be a major cause of cybrid lethality. [ABSTRACT FROM AUTHOR]

This study was conducted to establish pig embryonic stem (ES)-like cell lines from nuclear transfer blastocysts. A green fluorescent protein (GFP)-expressing cell line was used as the source of donor cells injected into the enucleated oocytes. Blastocysts were collected at D5 (the fifth day), D7 (the seventh day) and D9 (the ninth day). Differential staining was used to assay the viability and development of blastocysts from the 3 days. The number of inner cell mass (ICM) cells increased from 1.83 ± 0.8 (D5) to 5.37 ± 1.2 (D7) to 7.56 ± 1.5 (D9). The expression profiles of embryonic stem (ES) cell factors (OCT4, SOX2, KLF4 and c-MYC) correlated best with the undifferentiated ES state and were identified by qPCR. The expression of the four factors was increased from D5 to D7, whereas the expression decreased from D7 to D9. We tried to isolate ES-like cells from these embryos. However, ES-like cells from the D7 blastocysts grew slowly and expressed alkaline phosphatase. The cells from the D9 blastocysts grew rapidly but did not express alkaline phosphatase. ES-like cells were not isolated from the D5 blastocysts. These results show that the cells from the D7 embryos are pluripotent but grow slowly. The cells from the D9 embryos grow rapidly but start to lose pluripotency. [ABSTRACT FROM AUTHOR]

Intracytoplasmic sperm injection (ICSI) is a technique commonly used in clinical and research settings. In mouse oocytes, conventional ICSI has a poor survival rate caused by a high level of lysis. Cytochalasin B (CB) is a toxic microfilament-inhibiting agent that is known to relax the cytoskeleton and enhance the flexibility of oocytes. CB has been used widely in nuclear transfer experiments to improve the success rate of the micromanipulation, however information describing the use of CB in ICSI is limited. Here, we demonstrated that the addition of 5 μg/ml CB to the manipulation medium of ICSI procedure significantly improved the survival rate of the ICSI embryos (80.74% vs. 89.50%, p < 0.05), and that there was no harm for the in vitro or in vivo development. The birth rates and birth weights were not significantly different between the CB-treated and -untreated groups. Interestingly, the microfilaments of the ICSI embryos were almost undetectable immediately after CB treatment; however, they gradually re-appeared and had fully recovered to the normal level 2 h later. Moreover, CB did not disturb spindle rotation, second polar body formation or pronuclei migration, and had no effect on the microtubules. We thus conclude that ICSI manipulation in CB-containing medium results in significantly improved survival rate of mouse ICSI embryos, and that short-term treatment with CB during ICSI manipulation does not have adverse effects on the development of ICSI embryos. [ABSTRACT FROM AUTHOR]

Conformational changes at supramolecular interfaces are fundamentally coupled to binding activity, yet it remains a challenge to probe this relationship directly. Within the nuclear pore complex, this underlies how transport receptors known as karyopherins proceed through a tethered layer of intrinsically disordered nucleopor-in domains containing Phe-Gly (FG)-rich repeats (FG domains) that otherwise hinder passive transport. Here, we use nonspecific proteins (i.e., BSA) as innate molecular probes to explore FG domain conformational changes by surface plasmon resonance. This mathematically diminishes the surface plasmon resonance refractive index constraint, thereby providing the means to acquire and correlate height changes in a surface-tethered FG domain layer to Kap binding affinities in situ with respect to their relative spatial arrangements. Stepwise measurements show that FG domain collapse is caused by karyopherin ß1 (Kapßl) binding at low concentrations, but this gradually transitions into a reextension at higher Kapßl concentrations. This ability to self-heal is intimately coupled to Kapß1-FG binding avidity that promotes the maximal incorporation of Kapßl into the FG domain layer. Further increasing Kapßl to physiological concentrations leads to a "pileup" of Kapßl molecules that bind weakly to unoccupied FG repeats at the top of the layer. Therefore, binding avidity does not hinder fast transport per se. Revealing the biophysical basis underlying the form-function relationship of Kapß1-FG domain behavior results in a convergent picture in which transport and mechanistic aspects of nuclear pore complex functionality are reconciled. [ABSTRACT FROM AUTHOR]

Milk from dairy cows contains the protein ß-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG. One tandem construct was expressed in the mammary gland of an ovine BLG-expressing mouse model, resulting in 96% knockdown of ovine BLG in milk. Following this in vivo validation, we produced a transgenic calf, engineered to express these tandem miRNAs. Analysis of hormonally induced milk from this calf demonstrated absence of BLG and a concurrent increase of all casein milk proteins. The findings demonstrate miRNA-me-diated depletion of an allergenic milk protein in cattle and validate targeted miRNA expression as an effective strategy to alter milk composition and other livestock traits. [ABSTRACT FROM AUTHOR]

Abstract: We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1–3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1–3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques. [Copyright &y& Elsevier]

Abstract: The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos. [Copyright &y& Elsevier]

ABSTRACT At fertilization, the sperm triggers resumption from the arrest, extrusion of the second polar body and pronuclear formation, the events of which are collectively acknowledged as 'oocyte activation'. In all species up to date, oocyte activation requires a fertilization-associated increase in the intracellular concentration of calcium. Especially in mammals, the signal of intracellular calcium rise at fertilization consists of periodical rises, which are also referred to as calcium oscillations. Our recent results suggest that these calcium oscillations have an important role in not only oocyte activation but also development of mammals. Pigs are animals of great agricultural value and ones in which assisted reproductive techniques, including somatic cell nuclear transfer, to produce gene-modified pigs. Although reconstructed embryos require artificial activation stimuli which mimic fertilization-associated increase of intracellular calcium in the oocytes, it has been known that the developmental ability of the oocytes after artificial activation is low and the regimen seems to be required for improvement. Recently we focused on two molecules, phospholipase C zeta and inositol 1,4,5-triphosphate receptor which have important roles in regulation of calcium oscillations during fertilization in mammals, including pigs. In this review, we will discuss the present status and future perspective of molecular mechanisms during fertilization in pigs. [ABSTRACT FROM AUTHOR]

Herpesvirus capsids traverse the nuclear envelope (NE) by utilizing an unusual export pathway termed nuclear egress. In this process, the viral capsid is delivered into the perinuclear space (PNS), producing a vesicular intermediate after fission. After fusion with the outer nuclear membrane (ONM), the naked capsid is released into the cytosol. A recent study now suggests that this pathway might be an endogenous cellular pathway, co-opted by viruses, that serves to transport cellular cargo exceeding the size limit imposed by the nuclear pore complex (NPC). We propose that one function of this pathway is to transport nuclear protein aggregates to the cytosolic autophagy machinery. Our model has implications for our understanding of laminopathies and related diseases affecting proteins residing at the inner nuclear membrane (INM) and nuclear lamina. [ABSTRACT FROM AUTHOR]

Abstract: Linker histone variants are involved in regulation of chromosome organization and gene transcription; several subtypes are expressed in the maturing oocyte and developing embryo. In Xenopus and mice, the transition between linker histone variants occurred following nuclear transfer, and apparently contributed to donor nuclear reprogramming. To determine whether such linker histone replacement occurred after bovine nuclear transfer, red fluorescent protein (RFP) tagged H1e (somatic linker histone H1e) donor cells and Venus tagged H1foo eggs were created, enucleated eggs were injected with donor cells, and embryos were created by fusion. Using fluorescence microscopy, release of H1e in the donor nucleus, acquisition of H1foo by donor chromosomes, and the H1foo-to-H1e transition were observed in live cells. Linker histone replacement occurred more slowly in bovine than murine embryos. Low levels of diffuse red fluorescence (H1e) in the donor nucleus were detected 5 h after fusion, at which time green fluorescence (H1foo) had incorporated into donor chromosomes. However, complete replacement did not occur until 8 h after fusion. We concluded that the linker histone transition was sufficiently conserved among species, which provided further evidence regarding its important role in nuclear reprogramming. [Copyright &y& Elsevier]

Abstract: The objective was to use dual markers to accurately select genetically modified donor cells and ensure that the resulting somatic cell nuclear transfer kids born were transgenic. Fetal fibroblast cells were transfected with dual marking gene vector (pCNLF-ng) that contained the red-shifted variant of the jellyfish green fluorescent protein (LGFP) and neomycin resistance (Neo) markers. Cell clones that were G418-resistant and polymerase chain reaction-positive were subcultured for several passages; individual cells of the clones were examined with fluorescence microscopy to confirm transgenic integration. Clones in which every cell had bright green fluorescence were used as donor cells for nuclear transfer. In total, 86.7% (26/30) cell clones were confirmed to have transgenic integration of the markers by polymerase chain reaction, 76.7% (23/30) exhibited fluorescence, but only 40% (12/30) of these fluorescent cell clones had fluorescence in all cell populations. Moreover, through several cell passages, only 20% (6/30) of the cell clones exhibited stable LGFP expression. Seven transgenic cloned offspring were produced from these cells by nuclear transfer. Overall, the reconstructed embryo fusion rate was 76.6%, pregnancy rates at 35 and 60 days were 39.1% and 21.7%, respectively, and the offspring birth rate was 1.4%. There were no significant differences between nuclear transfer with dual versus a single (Neo) marker (overall, 73.8% embryo fusion rate, 53.8% and 26.9% pregnancy rates, and 1.9% birth rate with five offspring). In conclusion, the use of LGFP/Neo dual markers and an optimized selection procedure reliably screened genetically modified donor cells, excluded pseudotransgenic cells, and led to production of human lactoferrin transgenic goats. Furthermore, the LGFP/Neo markers had no adverse effects on the efficiency of somatic cell nuclear transfer. [Copyright &y& Elsevier]

Objectives This study compared rate of cell proliferation, viability, cell size, expression patterns of genes related to pluripotency and epigenetic modification between canine foetal fibroblasts (c FF) and canine adipose tissue-derived mesenchymal stem cells (cAd- MSC). Materials and methods Proliferation pattern, cell viability as well as cell size at each passage of c FF and cAd- MSC were measured when cultures reached confluence. In addition, real-time PCR was performed to investigate expression of Dnmt1, HDAC1, OCT4, SOX2, BAX, BCL2 genes with reference to β-actin gene expression as an endogenous control in both cell lines. Results c FF and cAd- MSC differed in number of generations, but not in doubling times, at all passages. Mean cell size of cAd- MSC was significantly smaller than that of c FF. Cell viability was significantly lower in c FFs and apoptotic level was significantly lower in cAd- MSC compared to passage-matched c FF. In the expression of genes related to pluripotency and epigenetic modification, level of HDAC1 in cAd- MSC was significantly higher than in c FF, but expression of Dnmt1 did not differ between the two groups. OCT4 and SOX2 were significantly more highly expressed in cAd- MSC compared to c FF. Conclusions cAd- MSC have higher stem-cell potential than c FF in terms of proliferation patterns, epigenetic modification and pluripotency, thus cAd- MSC could be more appropriate than c FF as donors of nuclei in somatic cell nuclear transfer for transgenesis. [ABSTRACT FROM AUTHOR]

Abstract: XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1–Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1. [Copyright &y& Elsevier]

As an abundant source that involves fewer ethical considerations, human abnormally fertilized zygotes are superior to oocytes as therapeutic cloning recipients of nuclear transfer. However, more effective manipulation conditions should be developed for somatic cell nuclear transfer (SCNT) studies using human abnormally fertilized zygotes as recipients. The present study found that the use of cytochalasin B was not necessary for, and even harmful to, the enucleation of human zygotes. This study also decreased the DNA methylation Levels in reconstructed embryos using a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), in an attempt to correct the abnormalities in DNA methylation that might play an important role in the failure of embryo development. After 5-aza-dC treatment and nuclear transfer (NT-Aza group), 32.7% of reconstructed embryos developed to the 8-cell stage, which is a much higher percentage than that of the nuclear transfer only (NT) group (11.1%). The DNA methylation level in the NT-Aza group was significantly lower than that of the NT group, as determined by 5-methylcytosine immunodetection. Based on the present results, this study recommends performing the enucleation procedure without cytochalasin B treatment and using 5-aza-dC in the culture of reconstructed embryos in human SCNT studies. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) is an alternative potential tool for the conservation of endangered. In this study, somatic cells were collected from a purebred 9-month-old male mastiff and an 11-month-old female mastiff. Oocytes that had been matured in vivo were retrieved from outbred dogs by laparoscopy. We used cycling cells as donor cells for SCNT. A total of 289 oocytes were reconstructed with each male or female somatic cell and then fused/activated simultaneously by electrical stimulation. Finally, 224 embryos were transferred to 16 recipients that had been synchronized naturally. The efficiency of delivery of cloned dogs (7.1%) was threefold higher than in previous reports. Moreover, one surrogate delivered four identical cloned female Tibetan Mastiff puppies; another three surrogates each delivered triplets. Microsatellite analysis demonstrated the genotypic identity of the cloned puppies. Thus, our study has demonstrated techniques that improve significantly the overall efficiency of SCNT in the canine species. [ABSTRACT FROM AUTHOR]

Contents Efficiency of the technique of somatic cell nuclear transfer critically depends on the cell cycle phase compatibility between the donor somatic cell nucleus and recipient cytoplasm. In this study, attempts were made to optimize conditions for cell cycle synchronization of bison ear fibroblasts at G0/G1 using different approaches such as using cells in confluency, after contact inhibition, serum starvation or treatment with dimethyl sulfoxide (DMSO) (0.5%, 1.0% and 2.0%), sodium butyrate (NaBu) (0.5, 1.0 and 2.0 m m), cytochalasin-B (CB) (7.5 μg/ml), cycloheximide (CHX) (7.5 μg/ml) and 6-dimethyl aminopurine (6-DMAP) (2.0 m m). A small piece of an ear of an adult female bison collected post-mortem 10 h after death was used for the preparation of fibroblast cells. The synchronization efficiency was determined by fluorescence-activated cell sorting. Higher proportion of G0/G1 phase was obtained when cells were subjected to serum starvation for 48 h (85.4%). Sodium butyrate had no effect on synchronization of cells at G0/G1 when the cells were treated for 24 and 48 h. Similarly, DMSO (0.5% and 1.0%) had also no effect on the proportion of cells at G0/G1 for 24 and 48 h. The synchronization ability of CB, CHX and 6-DMAP at G0/G1 phase was equally effective when cells were treated for 4 h (68.5%, 68.7% and 67.4%) and 24 h (67.8%, 66.1% and 67.5%). In conclusion, this study shows that cells subjected to serum starvation for 24-48 h or confluent monolayer, or cycling cells treated with 1.0% DMSO or 2.0 m m NaBu for 24 h showed best synchronization in G0/G1 phase of cell cycle. [ABSTRACT FROM AUTHOR]

Contents Embryo biotechnology has become one of the prominent high businesses worldwide. This technology has evolved through three major changes, that is, traditional embryo transfer ( in vivo embryo production by donor superovulation), in vitro embryo production by ovum pick up with in vitro fertilization and notably current cloning technique by somatic cell nuclear transfer and transgenic animal production. Embryo biotechnology has widely been used in dairy and beef cattle industry and commercial bovine embryo transfer has become a large international business. Currently, many developed biotechnologies during the period from early oocyte stage to pre-implantation embryos can be used to create new animal breeds and accelerate genetic progression. Based on recent advances in embryo biotechnologies and authors current studies, this review will focus on a description of the application of this technology to beef cattle improvement and discuss how to use this technology to accelerate beef cattle breeding and production. The main topics of this presentation include the following: (i) how to increase calf production numbers from gametes including sperm and oocyte; (ii) multiple ovulation and embryo transfer breeding schemes; (iii) in vitro fertilization and intracytoplasm sperm injection in bovine; (iv) pronuclear development and transgenic animals; (v) sex selection from sperm and embryos; (vi) cloning and androgenesis; (vii) blastocyst development and embryonic stem cells; (viii) preservation of beef cattle genetic resources; and (ix) conclusions. [ABSTRACT FROM AUTHOR]

Contents Somatic cells in milk are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important in animals that are susceptible to risks of bacterial infection on biopsy collection. In this study, a minimum of 10 milk samples were collected from each of the three buffaloes representing Murrah breed. All the samples were processed immediately and cell colonies were obtained. Cell colonies from one buffalo (MU-442) survived beyond 10 passages and were evaluated by fluorescence microscopy and used in nuclear transfer experiments. In culture, these cells expressed vimentin, indicating they were of fibroblast origin similar to ear cells. We compared the effectiveness of cloning using those milk-derived fibroblast (MDF) cells and fibroblast cells derived from the ear derived fibroblast (EDF). Fusion and cleavage rates of MDF-NT and EDF-NT embryos were found to be similar (92.43 ± 1.28% vs 94.98 ± 1.24%, and 80.27 ± 1.75% vs 84.56 ± 3.73%, respectively; p > 0.01); however, development to blastocyst stage and total cell number was higher for EDF-NT embryos (50.24 ± 2.54%, 227.14 ± 13.04, respectively, p < 0.01), than for MDF-NT embryos (16.44 ± 0.75%, 170.57 ± 4.50 respectively). We conclude that somatic cells from milk can be cultured effectively and used as nucleus donor to produce cloned blastocyst-stage embryos. [ABSTRACT FROM AUTHOR]

This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, “Human somatic cell nuclear transfer (cloning),” last published in Fertil Steril 2000;74:873-6. [ABSTRACT FROM AUTHOR]

Nuclear transfer (NT) remains the most effective method to reprogram somatic cells to totipotency. Somatic cell nuclear transfer (SCNT) efficiency however remains low, but recurrent problems occurring in partially reprogrammed cloned embryos have recently been identified and some remedied. In particular, the trophectoderm has been identified as a lineage whose reprogramming success has a large influence on SCNT embryo development. Several interspecific hybrid and cybrid reprogramming systems have been developed as they offer various technical advantages and potential applications, and together with SCNT, they have led to the identification of a series of reprogramming events and responsible reprogramming factors. Interspecific incompatibilities hinder full exploitation of cross-species reprogramming systems, yet recent findings suggest that these may not constitute insurmountable obstacles. [Copyright &y& Elsevier]

Pluripotent cells arise within the inner cell mass (ICM) of mammals and have the potential to generate all cell types of the adult organism through a process of commitment and ordered differentiation. Despite many decades of investigation, the mechanisms that guide and stabilise cell fate choice as well as those that can be engineered to promote its reversal, remain only partially resolved. Reprogramming of somatic cells towards a pluripotent-like state can be achieved by several different experimental routes including nuclear transfer, the supply of a defined cocktail of transcription factors, or by fusing somatic cells with a pluripotent stem cell partner. These approaches have been used to demonstrate the remarkable intrinsic epigenetic plasticity of many terminally differentiated cell types, as well as to define the factors that are required for pluripotent conversion. In this review we summarise some recent advances using cell fusion-based studies to better understand the basis of pluripotency and the epigenetic mechanisms that promote cell type inter-conversion. [Copyright &y& Elsevier]

Abstract: The fate of foreign mitochondrial DNA (mtDNA) following somatic cell nuclear transfer (SCNT) is still controversial. In this study, we examined the transmission of the heteroplasmic mtDNA of gaur donor cells and recipient bovine oocytes to an offspring and aborted and mummified fetuses at various levels during the development of gaur–bovine interspecies SCNT (iSCNT) embryos. High levels of the donor cell mtDNA were found in various tissue samples but they did not have any beneficial effect to the survival of iSCNT offspring. However, the factors on mtDNA inheritance are unique for each iSCNT experiment and depend on the recipient oocyte and donor cell used, which might play an important role in the efficiency of iSCNT. [Copyright &y& Elsevier]

BACKGROUND Fetal cells (microchimerism) are acquired by women during pregnancy. Fetal microchimerism persists decades later and includes cells with pluripotent capacity. Persistent microchimerism has the capacity for both beneficial and detrimental maternal health consequences. Both miscarriage and termination of pregnancy can result in fetal microchimerism. We sought to determine whether cellular fetal microchimerism is acquired during management of pregnancy loss and further explored factors that could influence fetal cell transfer, including viability of fetal tissue, surgical versus medical management and gestational age. METHODS Pregnant women (n= 150 samples from 75 women) with singleton pregnancies undergoing a TOP (n= 63) or treatment for embryonic or fetal demise (miscarriage, n= 12) were enrolled. Mononuclear cells were isolated from blood samples drawn before, and 30 min after, treatment. Fetal cellular microchimerism concentrations were determined using quantitative PCR for a Y chromosome-specific sequence, expressed as genome equivalents of fetal DNA per 100 000 maternal cell equivalents (gEq/105). Detection rate ratios were determined according to clinical characteristics. RESULTS Cellular fetal microchimerism was found more often in post- compared with pretreatment samples, 24 versus 5% (P= 0.004) and at higher concentrations, 0–36 versus 0–0.7 gEq/105 (P< 0.001). Likelihood of microchimerism was higher in surgical than medical management, detection rate ratio 24.7 (P= 0.02). The detection rate ratio for TOP versus miscarriage was 16.7 for known male fetuses (P= 0.02). Microchimerism did not vary with gestational age. CONCLUSIONS Significant fetal cell transfer occurs during miscarriage and TOP. Exploratory analyses support relationships between obstetric clinical factors and acquisition of fetal cellular microchimerism; however, our limited sample size precludes definitive analysis of these relationships, and confirmation is needed. In addition, the long-term persistence and potential consequences of fetal microchimerism on maternal health merit further investigation. [ABSTRACT FROM AUTHOR]

Abstract: This study was conducted to evaluate the efficiency of potassium simplex optimization medium with amino acids (KSOMaa) as a basal culture medium for caprine intraspecies somatic cell nuclear transfer (SCNT) and caprine-bovine interspecies somatic cell nuclear transfer (iSCNT) embryos. The effect of increased glucose as an energy substrate for late stage development of cloned caprine embryos in vitro was also evaluated. Enucleated caprine and bovine in vitro matured oocytes at metaphase II were reconstructed with caprine ear skin fibroblast cells for the SCNT and iSCNT studies. The cloned caprine and parthenogenetic embryos were cultured in either KSOMaa with 0.2 mM glucose for 8 days (Treatment 1) or KSOMaa for 2 days followed by KSOMaa with additional glucose at a final concentration of 2.78 mM for the last 6 days (Treatment 2). There were no significant differences in the cleavage rates of SCNT (80.7%) and iSCNT (78.0%) embryos cultured in KSOMaa medium. Both Treatment 1 and Treatment 2 could support in vitro development of SCNT and iSCNT embryos to the blastocyst stage. However, the blastocyst development rate of SCNT embryos was significantly higher (P < 0.05) in Treatment 2 compared to Treatment 1. Increasing glucose for later stage embryo development (8-cell stage onwards) during in vitro culture (IVC) in Treatment 2 also improved both caprine SCNT and iSCNT embryo development to the hatched blastocyst stage. In conclusion, this study shows that cloned caprine embryos derived from SCNT and iSCNT could develop to the blastocyst stage in KSOMaa medium supplemented with additional glucose (2.78 mM, final concentration) and this medium also supported hatching of caprine cloned blastocysts. [Copyright &y& Elsevier]

Abstract: Domesticated animals cloned by somatic cell nuclear transfer (SCNT) generally have poor developmental competency, with many developmental abnormalities attributed to incomplete reprogramming of the nuclear genome and abnormal expression of genes important for regulation of growth and development. To investigate the molecular mechanism leading to the abnormalities of cloned animals, pathologic and histologic analyses were conducted on seven cloned cattle that were oversized at birth and had cardiac and pulmonary abnormalities. Quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis of four imprinted genes IGF2, IGF2R, H19, and GRB10, as well as genes from related regulatory networks, were performed in liver, lung, kidney, and muscle to investigate disruption of expression. Expression of IGFBP2 was not detected in morphologically normal cloned cattle, but was detected in the liver, lung, kidney, and thymus of abnormal calves. Expression levels of IGF1 and imprinted genes IGF2 and H19 were substantially higher in these organs of abnormal cattle. In contrast, expression levels of GRB10, CTSD, and TRPV2 were substantially lower in abnormal cattle. Transcript abundance of IGFBP6 was higher in kidney, but lower in liver and lung. In conclusion, we inferred that altered expression of imprinted and related genes may be closely related to increased birth weight and pathologic changes in abnormal cloned cattle. [Copyright &y& Elsevier]

Cellular stresses significantly affect nuclear transport systems. Nuclear transport pathways mediated by importin β-family members, which are active under normal conditions, are downregulated. During thermal stress, a nuclear import pathway mediated by a novel carrier, which we named Hikeshi, becomes active. Hikeshi is not a member of the importin β family and mediates the nuclear import of Hsp70s. Unlike importin β family-mediated nuclear transport, the Hikeshi-mediated nuclear import of Hsp70s is not coupled to the GTPase cycle of the small GTPase Ran but rather is coupled with the ATPase cycle of Hsp70s. Hikeshi-mediated nuclear import is essential for the attenuation and reversal of the thermal stress response in human cells. The mechanism and functions of this newly identified nuclear import pathway will be discussed. [ABSTRACT FROM AUTHOR]

Successful gene therapy asks for multifunctional vectors which can not only protect DNA from degradation but also transfer it into nuclear and subsequently express the loaded gene. Here we reported a novel multilayered delivery system constructed with DNA, protamine (Pro) and polyethylenimine (PEI) via lay-by-layer (LbL) technique, which posed multifunctions. DNA was previously condensed into a compact core with Pro which also contained nuclear localisation signals (NLS) domains for nuclear transfer. Then additional DNA was deposited as the first layer onto the cationic core via the electrostatic attraction which would increase the loading dose of DNA. At last, PEI was absorbed as the outmost layer to achieve the endosomal escape. Therefore a quaternary polyplexes which offered high loading of DNA, nuclear transfer ability and endosomal escape capability was constructed with the LbL technique. The obtained quaternary polyplexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA protection capability. Compared with commercially available PEI/DNA complexes, the novel multifuctional vector exhibited not only lower cytotoxicity (P<0.05) but also higher transfection efficiency in HepG2 and HeLa cells (P<0.05) in vitro test. [ABSTRACT FROM AUTHOR]

There is ongoing debate about whether it is ethically acceptable to allow the creation of cloned embryos in order to produce human stem cells. A cloned embryo is created through a process called somatic-cell nuclear transfer, often known as ‘therapeutic’ cloning. The value of stem cells lies in their capacity to become any sort of cell in the human body. This capacity is particularly useful for treating medical conditions where stem cells can be used to repair or replace damaged tissue. The value of a cloned embryo is that the DNA from a person who needs stem cell treatment can be used to create the clone and thereby minimise the likelihood that any inserted stem cells will be rejected. Research on the use of cloned stem cells is in its earliest stage. [ABSTRACT FROM AUTHOR]

Epigenetic reprogramming can be achieved in different ways, including nuclear transfer, cell fusion, or the expression of transcription factors (TFs). Combinatorial overexpression provides an opportunity to define the minimal core network of TFs that instructs specific cell fates. This approach has been employed to induce mouse and human pluripotency and differentiated cell types from cells that can be also as distant as cells from different germ layers. This suggests the possibility that any specific cell type may be directly converted into another if the appropriate reprogramming TF core is determined. Herein, we review the factors used for reprogramming multiple cell identities and raise the question of whether there is a common underlying blueprint for reprogramming factors. In addition to the generation of human cell types of interest for cell-replacement therapies, we propose that the TF-mediated conversion of differentiated cell types, especially somatic stem cells, will have an impact on our understanding of their biological development. [ABSTRACT FROM AUTHOR]

We used somatic cell nuclear transfer (SCNT) to generate a mouse from the nucleus of an lgG1+ ovalbumin-specific B cell. The resulting OBI mice show generally normal B-cell development, with elevated percentages of marginal zone B cells and a reduction in B-1 B cells. Whereas OBI RAG1-/- mice have exclusively lgG1 anti-ovalbumin in their serum, OBI mice show elevated levels of anti-ovalbumin of nearly all isotypes 3' of the γ1 constant region in the IgH locus, indicating that class switch recombination (CSR) occurs in the absence of immunization with ovalbumin. This CSR is associated with the presence of lgM+lgG1+ double producer B cells that represent <1 % of total B cells, accumulate in the peritoneal cavity, and account for near-normal levels of serum IgM and lgG3. [ABSTRACT FROM AUTHOR]

Abstract: Several research groups have suggested that the embryonic–abembryonic (Em–Ab) axis in the mouse can be predicted by the first cleavage plane of the early embryo. Currently, it is not known whether this early patterning occurs in cloned embryos produced by nuclear transfer and whether it affects development to term. In this work, the relationship between the first cleavage plane and the Em–Ab axis was determined by the labeling of one blastomere in cloned mouse embryos at the 2-cell stage, followed by ex-vivo tracking until the blastocyst stage. The results demonstrate that approximately half of the cloned blastocysts had an Em–Ab axis perpendicular to the initial cleavage plane of the 2-cell stage. These embryos were classified as “orthogonal” and the remainder as “deviant”. Additionally, we report here that cloned embryos were significantly more often orthogonal than their naturally fertilized counterparts and overexpressed Sox2. Orthogonal cloned embryos demonstrated a higher rate of post-implantation embryonic development than deviant embryos, but cloned pups did not all survive. These results reveal that the angular relationship between the Em–Ab axis and the first cleavage plane can influence later development and they support the hypothesis that proper early patterning of mammalian embryos is required after nuclear transfer. [Copyright &y& Elsevier]

Abstract: Somatic cell nuclear transfer (SCNT) has been established for the transmission of specific nuclear DNA. However, the fate of donor mitochondrial DNA (mtDNA) remains unclear. Here, we examined the fate of donor mtDNA in recloned pigs through third generations. Fibroblasts of recloned pigs were obtained from offspring of each generation produced by fusion of cultured fibroblasts from a Minnesota miniature pig (MMP) into enucleated oocytes of a Landrace pig. The D-loop regions from the mtDNA of donor and recipient differ at nucleotide sequence positions 16050 (A→T), 16062 (T→C), and 16135 (G→A). In order to determine the fate of donor mtDNA in recloned pigs, we analyzed the D-loop region of the donor’s mtDNA by allele-specific PCR (AS-PCR) and real-time PCR. Donor mtDNA was successfully detected in all recloned offspring (F1, F2, and F3). These results indicate that heteroplasmy that originate from donor and recipient mtDNA is maintained in recloned pigs, resulting from SCNT, unlike natural reproduction. [Copyright &y& Elsevier]

Contents A recently emerged concept utilizing a controlled environmental impact as a treatment for cells and tissues aims to improve neither the in vitro conditions nor the procedures, but the cell itself. Hydrostatic pressure stress emerged as the most controllable and most effective stressor, proving the principle that controlled stress improves cell performance in in vitro procedures, whereas further studies using different stressors (osmotic, oxidative or mechanic stresses) supported the principle. The present summary reviews studies of various stress treatments to treat oocytes of three species (murine, porcine, human) before vitrification, in vitro maturation, enucleation and somatic cell nuclear transfer. Eventually, cleavage and blastocyst rates and - in cases when hydrostatic pressure was used - blastocyst cell number and birth rates as well were significantly improved compared to untreated controls. [ABSTRACT FROM AUTHOR]

Contents The aim of this article is to review dog cloning research and to suggest its applications based on a discussion about the normality of cloned dogs. Somatic cell nuclear transfer was successfully used for production of viable cloned puppies despite limited understanding of in vitro dog embryo production. Cloned dogs have similar growth characteristics to those born from natural fertilization, with no evidence of serious adverse effects. The offspring of cloned dogs also have similar growth performance and health to those of naturally bred puppies. Therefore, cloning in domestic dogs can be applied as an assisted reproductive technique to conserve endangered species, to treat sterile canids or aged dogs, to improve reproductive performance of valuable individuals and to generate disease model animals. [ABSTRACT FROM AUTHOR]

Abstract: To determine whether the nuclei of early growing stage porcine oocytes can mature to the MII stage, we examined meiotic competence of nuclei that had been fused with enucleated GV oocytes using the nuclear transfer method. In vitro matured oocytes were enucleated and then fused with early growing oocytes (30–40 μm in diameter) from 5 to 7-wk-old piglets using the hemagglutinating virus of Japan (HVJ). Reconstructed oocytes were cultured for 24 h to the MII stage. Although these oocytes extruded the first polar body, they did not contain normal haploid chromosomes, and the spindles were misaligned or absent at the metaphase II (MII) stage. Furthermore, maturation promoting factor (MPF) activity levels were low in oocytes reconstructed with early growing oocytes at metaphase I (MI) and MII. In contrast, mitogen-activated protein kinase (MAPK) activity was detected between the MI and MII stages, although at slightly lower levels. In conclusion, the nuclei of early growing oocytes did not accomplish normal meiotic division in matured oocytes due to misaligned or absent spindle formation. [Copyright &y& Elsevier]

Purpose: This study compared the expression of genes involved in pluripotency, segregation of inner cell mass (ICM) and trophectoderm (TE), and primitive endoderm (PE) formation in porcine embryos produced by in vitro fertilization (IVF), parthenogenetic activation (PA), and nuclear transfer (NT) using either fetal fibroblasts (FF-NT) or mesenchymal stem cells (MSC-NT). Methods: Blastocyst formation and total cell number were analyzed. The expression patterns of transcripts, including SRY-related HMG-box gene 2 (SOX2), reduced expression gene 1 (REX1/ZFP42), LIN28, caudal type homeobox 2 (CDX2), TEA domain family member 4 (TEAD4), integrin beta 1 (ITGB1) and GATA6 were assessed at the 4-8 cell and blastocyst stage embryos by real-time PCR. Results: Developmental rates to blastocyst stage and total cell number were higher in IVF and PA embryos than in NT embryos. But MSC-NT embryos had increased blastocyst formation and higher total cell number compared to FF-NT embryos. The relative expressions of transcripts were higher in blastocysts than in 4-8 cell stage embryos. The mRNA expression levels of SOX2 and REX1 were largely similar in embryos of different origins. However, the genes such as LIN28, CDX2, TEAD4, ITGB1 and GATA6 showed the differential expression pattern in PA and NT embryos compared to IVF embryos. Importantly, the transcript levels in MSC-NT embryos were relatively less variable to IVF than those in FF-NT embryos. Conclusion: MSCs seem to be better donors for porcine NT as they improved the developmental competency, and influenced the expression pattern of genes quite similar with IVF embryos than that of FFs. [ABSTRACT FROM AUTHOR]

Contents The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p < 0.05) and survival rate (75.7% vs 53.3%, p < 0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p < 0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p < 0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p < 0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development. [ABSTRACT FROM AUTHOR]

Contents Cloned rabbits have been produced for many years by somatic cell nuclear transfer (SCNT). The efficiency of cloning by SCNT, however, has remained extremely low. Most cloned embryos degenerate in utero, and the few that develop to term show a high incidence of post-natal death and abnormalities. The cell type used for donor nuclei is an important factor in nuclear transfer (NT). As reported previously, NT embryos reconstructed with fresh cumulus cells (CC-embryos) have better developmental potential than those reconstructed with foetal fibroblasts (FF-embryos) in vivo and in vitro. The reason for this disparity in developmental capacity is still unknown. In this study, we compared active demethylation levels and morphological changes between the nuclei of CC-embryos and FF-embryos shortly after activation. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized and cloned rabbit embryos revealed that there was no detectable active demethylation in rabbit zygotes or NT-embryos derived from either fibroblasts or CC. In the process of nuclear remodelling, however, the proportion of nuclei with abnormal appearance in FF-embryos was significantly higher than that in CC-embryos during the first cell cycle. Our study demonstrates that the nuclear remodelling abnormality of cloned rabbit embryos may be one important factor for the disparity in developmental success between CC-embryos and FF-embryos. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear-transferred (SCNT) oocytes have a high potential for development in vitro, but a large proportion of embryos that are transferred to recipients is aborted before parturition. The precise mechanism for the high abortion rate is unknown, but abnormal placenta formation is frequently observed in SCNT-cloned pregnancies. The present study examined the effects of treating the recipients with cyclosporin A (CsA), an immunoprotectant, on the proportion of fetuses resulting from SCNT-cloned pregnancies. Cloned embryos developed from enucleated oocytes and receiving cumulus cells from F1 (C57BL/6 × DBA, H-2b/d) females were transferred to outbred ICR (in which the H-2 complex was not fixed) recipient females. Each recipient received an intraperitoneal injection of CsA or vehicle. Compared with vehicle, administration of CsA to recipients on day 4.5 of pregnancy significantly increased the proportion of fetuses observed on day 10.5. The proportion of fetuses at day 18.5 of pregnancy in recipients receiving CsA treatment was slightly higher than that in controls. This study is the first to report that CsA administration increases the proportion of fetuses resulting from SCNT-cloned pregnancies. [ABSTRACT FROM PUBLISHER]

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos. [ABSTRACT FROM PUBLISHER]

Somatic cells derived from zebrafish haploid larval (both androgenetic and gynogenetic) cultures were used as donors for nuclear transplant into non-enucleated oocytes. Nuclei were transplanted either before or simultaneously with oocyte activation in the central region and in the incipient animal pole, respectively. Against expected results, 20% of transplanted embryos during oocyte activation using cells of gynogenetic origin reached the 100% epiboly stage, even two survived for up to 5 days, whereas no development was observed when cells from androgenetic origin were used. Results derived from this work open a novel possibility of studying somatic cell reprogramming and imprinting phenomena in zebrafish. [ABSTRACT FROM PUBLISHER]

Somatic cell nuclear transfer allows the generation of cloned embryonic stem cells (ESC) and cloned mice from natural killer T (NKT) cells, an innate-type invariant T cell. The progeny of these cloned mice harboring a rearranged T cell receptor α loci specific for NKT cells, Vα14-Jα18, possess an increased number of NKT cells in the primary as well as in the secondary lymphoid organs. NKT cells in these mice are able to produce interferon-γ (IFN-γ) and interleukin-4 (IL-4) when stimulated with dendritic cells (DC) pulsed with an NKT cell agonist, α-galactosylceramide (α-GalCer). The directed differentiation of cloned ESC toward T cells results in quasi-exclusive generation of NKT cells. These NKT cells are functional as evidenced by the production of cytokines such as IFN-γ, IL-4, IL-10, and IL-13 in response to α-GalCer. Furthermore, they mature autonomously in vivo upon adoptive transfer, and exhibit an antigen-specific adjuvant effect, resulting in IFN-γ production from CD8+ T cells. This effect is evident when the growth of tumors is inhibited in an antigen-dependent manner upon tumor inoculation into the mouse. Unfortunately, in humans, NKT cells are rare, and there is no guarantee that the same technique will be applicable for use in the clinic. We, therefore, exploited a different type of T cells in humans. We established induced pluripotent stem cells (iPSC) from these T cells, and succeeded in directed differentiation of the iPSC into monoclonal T cells. The availability of iPSC-derived monoclonal T cells paves the way for their use in regenerative medicine. In addition, they will be useful for drug screening to target unmet medical needs such as autoimmunity, severe infection, allergy, and cancer. [ABSTRACT FROM AUTHOR]

The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived. [ABSTRACT FROM AUTHOR]