Donor Nuclei Research Articles

Cytoplasmic volume of recipient oocytes affects the nucleus reprogramming and the developmental competence of HMC buffalo (Bubalus bubalis) embryos.

The present study was undertaken to examine the effects of cytoplasmic volume on nucleus reprogramming and developmental competence of buffalo handmade cloning (HMC) embryos. We found that both HMC embryos derived from ~150% cytoplasm or ~225% cytoplasm resulted in a higher blastocyst rate and total cell number of blastocyst in comparison with those from ~75% cytoplasm (25.4 ± 2.0, 27.9 ± 1.6% vs. 17.9 ± 3.1%; 150 ± 10, 169 ± 12 vs. 85 ± 6, P<0.05). Meanwhile, the proportions of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) were also increased in the embryos derived from ~150 or ~225% enucleated cytoplasm compared to those from ~75% cytoplasm. Moreover, HMC embryos derived from ~225% cytoplasm showed a decrease of global DNA methylation from the 2-cell to the 4-cell stage in comparison with those of ~75% cytoplasm (P<0.05). Furthermore, the expression of embryonic genome activation (EGA) relative genes (eIF1A and U2AF) in HMC embryos derived from ~225% cytoplasm at the 8-cell stages was also found to be enhanced compared with that of the ~75% cytoplasm. Two of seven recipients were confirmed to be pregnant following transfer of blastocysts derived from ~225% cytoplasm, and one healthy cloned calf was delivered at the end of the gestation period, whereas no recipients were pregnant after the transfer of blastocysts derived from ~75% cytoplasm. These results indicate that the cytoplasmic volume of recipient oocytes affects donor nucleus reprogramming, and then further accounted for the developmental ability of the reconstructed embryos.

35 Interspecies Somatic Cell Nuclear Transfer Embryos that Form Nucleoli do not Always Activate Mitochondrial Functional Differentiation at the Time of Embryonic Genome Activation

Embryonic genome activation (EGA) is a complex process that needs a good orchestration of all biochemical processes at the time of maternal-to-embryonic transition. Mitochondria are strictly dependent on the nucleus for their correct activity as ~1500 mitochondrial genes have nuclear localisation. The finding of transcriptional activation and accumulation of mRNAs related to mitochondrial biogenesis (Mtango et al. 2008 Reprod. Fertil. Dev. 20, 846-859) around the time of EGA confirmed the role of nucleus in this process. Studying mitochondria behaviour in interspecies somatic cell nuclear transfer (iSCNT) embryos (Lagutina et al. 2010 Reproduction 140, 273-285), we have found that at the time of EGA, mitochondria activation could be demonstrated by JC-1 accumulation. We suggested that comparison of the mean green fluorescence intensity (FI) that corresponds to the fluorescence of the monomeric form of the dye and correlates to relative mitochondrial mass (Mancini et al. 1997) in iSCNT and control nuclear transfer (NT) could serve as a test to assess EGA in iSCNT embryos. The aim of this study was to estimate nuclear-cytoplasmic interaction in iSCNT embryos that formed nucleoli at the time of EGA (Lagutina et al. 2011 Reproduction 141, 453-465) such as embryos derived from bovine oocytes and bovine (control), buffalo or ovine donor nuclei, and from porcine oocytes and porcine (control), horse, or rabbit nuclei. Embryos 72 h after activation were stained with 2 μM JC-1 in SOF-HEPES with 10% FCS at 37°C for 1 h. Images were collected using a fluorescein isothiocyanate (FITC) filter and analysed with Adobe Photoshop Elements 2 (Adobe Systems, San Jose, CA, USA). The data are presented as mean FI of the embryo. To demonstrate the effect of EGA inhibition on mitochondria, bovine and porcine NT embryos were cultured in medium supplemented with 25 μg/mL α-amanitin (AA) from 48 to 72 h after activation. The analyses of mean FI of the embryos showed that ovine and buffalo nuclei were able to support mitochondrial mass accumulation in iSCNT embryos with bovine oocytes equal to control bovine NT embryos (35 ± 11.2; 41.9 ± 14.8; 36.2 ± 7.6, respectively) that was significantly higher than in bovine embryos treated with AA (15.4 ± 4.9; P &lt; 0.05). In the iSCNT embryos composed of porcine oocytes and equine or rabbit nuclei, mean FI values (20 ± 13.4; 18.3 ± 5.5, respectively) were comparable with those in porcine NT embryos treated with AA (16.2 ± 6.2), and were significantly lower than in porcine control (91 ± 47.7; P &lt; 0.05) NT embryos, demonstrating the inability of equine and rabbit nuclei to properly govern the porcine mitochondria mass growth at the time of EGA. In conclusion, nucleolus formation and activation of nuclear encoded mitochondrial genes at the time of EGA cannot serve, per se, as a marker of correct embryonic genome activation in iSCNT embryos, because, in our conditions, no iSCNT embryos developed to blastocyst (Lagutina et al. 2010 Reproduction 140, 273-285). This knowledge about behaviour of different embryo compartments at the time of EGA could extend our understanding of the whole process. This work was funded by Translink (EU FP7 no. 603049) and Xenoislet projects (EU FP7 no. 601827).

SV40 Large T Antigen Disrupts Embryogenesis of Canine and Porcine Somatic Cell Nuclear Transfer Embryo

BackgroundSomatic cell nuclear transfer (SCNT) is a useful biotechnological tool for transgenic animal production using genetically modified somatic cells (GMSCs). However, there are several limitations preventing successful transgenic animal generation by SCNT, such as obtaining proper somatic donor cells with a sufficiently long life span and proliferative capacity for generating GMSCs. Here, we established simian virus 40 large T antigen (SV40LT)-mediated lifespan-extended canine fibroblast cells (SV40LT-K9 cells) and evaluated their potential as nuclei donors for SCNT, based on cellular integrity and SCNT embryo development.ResultsSV40LT did not cause canine cell transformation, based on cell morphology and proliferation rate. No anchorage-independent growth in vitro and tumorigenicity in vivo were observed. After SCNT with SV40LT-K9 cells, embryos were transferred into surrogate dogs. All dogs failed to become pregnant. Most embryos did not proceed past the 8-cell stage and only one surrogate showed an implantation trace in its oviduct, indicating that the cells rarely developed into blastocysts. Because of the absence of an in vitro maturation method for canine embryos, we performed identical experiments using porcine fibroblast cells. Similarly, SV40LT did not transform porcine fibroblast cells (SV40LT-Pig cells). During in vitro development of SV40LT-Pig cell-driven SCNT embryos, their blastocyst formation rate was clearly lower than those of normal cells. Karyotyping analysis revealed that both SV40LT-K9 and SV40LT-Pig cells had aberrant chromosomal statuses.ConclusionsAlthough lifespan-extended canine and porcine cells via SV40LT exhibit no apparent transforming changes, they are inappropriate for use as nuclei donors for SCNT because of their aneuploidy.

Cytoplasmic Effects on the Agronomic and Physiological Traits of Dual‐Purpose Genic Male Sterile Substitution Lines of Rice

The two‐line hybrid rice (Oryza sativa L.) system is an effective way for breeding and developing super rice. However, few studies have been conducted on the cytoplasmic effects. In this study, the sterile ZhunS substitution lines were acquired by using ZhunS as the nucleus donor, and rice cultivars from indica, temperate japonica, and tropical japonica subgroups and wild species were used as cytoplasmic donors. The agronomic and physiological traits were analyzed. The results indicated that the cytoplasms and ZhunS nuclei had interaction. The shoot dry weight, grain number per panicle, grain weight per plant, and net photosynthesis rate of substitution line with the cytoplasm from Chaling common wild rice (‘Chaling CWR’) were higher than those of ZhunS, whereas the most of these traits in the substitution line with the cytoplasm from tropical japonica rice ‘Lemont’ were lower. When the substitution lines were crossed with parents, the grain yield of a hybrid of Chaling CWR//ZhunS and 9311 was increased by 5.1% as compared with that of the hybrid between ZhunS and 9311. Therefore, Chaling CWR//ZhunS was an efficient sterile line parent. The entire growth periods of the hybrid combinations of Lemont//ZhunS were shortened by &gt;4 d, thus Lemont//ZhunS could be used as an early‐season line. The results provided new information for the development of novel cytoplasmic sources and promotion of heterosis in two‐line hybrid rice.

Effect of POU5F1 Expression Level in Clonal Subpopulations of Bovine Fibroblasts Used as Nuclear Donors for Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) success is partially hindered by the low epigenetic reprogramming efficiency of the donor cell. Previous studies suggest cellular heterogeneity among donor nuclei in regard to reprogramming potential, which precludes comparison among different strategies to increase cloning success. In this context, we evaluated the effect of using clonal cell populations (CPs) of bovine adult fibroblasts established by single-cell plating in SCNT. Different CPs were evaluated in regard to proliferation rate, senescence level, and chromosome stability, as well as for POU5F1 (POU class 5 homeobox 1) mRNA expression levels. In total, 9 of 24 CPs (37.5%) were successfully expanded in vitro up to the fourth passage and shown to proliferate following cryopreservation, at which time cell analyses were performed. The use of a CP with low senescence level, normal karyotype, and highest POU5F1 expression levels did not improve embryo development rates or quality following SCNT. As previously suggested, this study supports the notion that levels of POU5F1 expression in the donor nucleus do not impact the SCNT results. Notably, the single-cell seeding approach used herein to isolate CPs may be extended to the evaluation of additional predictor markers of reprogrammability success for SCNT in future experiments.

Cloning Mice.

Viable and fertile mice can be generated by somatic nuclear transfer into enucleated oocytes, presumably because the transplanted somatic cell genome becomes reprogrammed by factors in the oocyte. The first somatic cloned offspring of mice were obtained by directly injecting donor nuclei into recipient enucleated oocytes. When this method is used (the so-called Honolulu method of somatic cell nuclear transfer [SCNT]), the donor nuclei readily and completely condense within the enucleated metaphase II-arrested oocytes, which contain high levels of M-phase-promoting factor (MPF). It is believed that the condensation of the donor chromosomes promotes complete reprogramming of the donor genome within the mouse oocytes. Another key to the success of mouse cloning is the use of blunt micropipettes attached to a piezo impact-driving micromanipulation device. This system saves a significant amount of time during the micromanipulation of oocytes and thus minimizes the loss of oocyte viability in vitro. For example, a group of 20 oocytes can be enucleated within 10 min by an experienced operator. This protocol is composed of seven parts: (1) preparing micropipettes, (2) setting up the enucleation and injection micropipettes, (3) collecting and enucleating oocytes, (4) preparing nucleus donor cells, (5) injecting donor nuclei, (6) activating embryos and culturing, and (7) transferring cloned embryos.

Effects of cytoplasts from Taiwan native yellow cattle on the cellular antioxidant ability of cloned Holstein cattle and their offspring

We previously demonstrated that the cellular thermotolerance of cloned cattle produced by combination of ooplasm (o) derived from Taiwan native yellow cattle (Y) and the donor (d) nucleus derived from Holstein (H) cattle (Yo-Hd) transmits to their offspring (Yo-Hd-F1). In the present study, the responses of mitochondria in these cloned cattle and their offspring after heat shock were investigated to elucidate influence of cytoplasmic input (i.e., ooplasm) during the course of heat stress. After heat shock, oxidative phosphorylation proteins (Complex III and IV) of ear fibroblast cells with Y-originated cytoplasm (including Y, Yo-Hd, and Yo-Hd-F1 cattle) were significantly greater (P < 0.05) than those of ear fibroblast cells with H-originated cytoplasm (including H, Ho-Hd, and Ho-Hd-F1 cattle). However, the expressions of Complex I and Complex II protein in heat shocked cells derived from Yo-Hd-F1 cattle were significantly (P < 0.05) higher than those of cell derived from cattle with the H-cytoplasm. The catalase (CAT) expression in heat shocked ear fibroblast cells derived from Yo-Hd and Yo-Hd-F1 cattle were significantly (P < 0.05) higher than that of cells derived from Ho-Hd-F1 cattle. However, the level of glutathione peroxidase (GPx) expression was higher (P < 0.05) in ear fibroblast cells with Y-originated cytoplasm compared to cells with H-originated cytoplasm. In conclusion, the expression of proteins involved in mitochondrial oxidative phosphorylation and antioxidant enzymes after heat shock was increased in ear fibroblast cells from cattle with Y-originated cytoplasm, which can be transmitted to their offspring.

Differential expression pattern of key regulatory developmental genes in pre-implant zona free cloned vs in vitro fertilized goat embryos

The success of Somatic cell nuclear transfer (SCNT) primarily depends on the extent of reprogramming of donor cells genome. The error of reprogramming may lead to inappropriate expression of embryonic genes at any stage of development. Under the present study the relative expression of different genes related to pluripotency (Oct-4 and Nanog), growth factors (IGF-2 and IGF-2R) and DNA methyltransferase gene (Dnmt-1) was evaluated in SCNT embryos at 8–16 cells, morula and blastocyst stages as compared to IVF group. In SCNT, significantly higher degree of relative expression was observed for Oct-4 in morula (1.41) and blastocysts (1.14) as compared to 8–16 cells (referral stage) whereas in IVF, a lower expression was observed at morula (0.82) stage. The expression of Nanog in SCNT embryos was increased significantly in morula (2.23) and decreased subsequently in blastocyst (0.56), whereas it was increased significantly from 8 to 16 cells to morula (1.62) and blastocyst (4.5) of IVF group. The IGF-2 and IGF-2R showed significantly higher expression rates in morula and blastocysts of SCNT (6.56, 5.90 and 1.11, 1.4) and IVF (8.69, 8.25 and 2.96, 3.91) embryos, respectively as compared to referral stage. The expression of Dnmt-1 was significantly higher in SCNT morula (1.29) and blastocyst (1.15) however in IVF, it was similar in 8–16 cells stage and morula but, higher in blastocyst (1.58). The dissimilar pattern of gene expression of SCNT might be a consequence of incomplete reprogramming of donor nucleus which resulted into lower blastocyst rate of SCNT as compared to IVF embryos.

Proper reprogramming of imprinted and non‐imprinted genes in cloned cattle gametogenesis

Epigenetic abnormalities in cloned animals are caused by incomplete reprogramming of the donor nucleus during the nuclear transfer step (first reprogramming). However, during the second reprogramming step that occurs only in the germline cells, epigenetic errors not corrected during the first step are repaired. Consequently, epigenetic abnormalities in the somatic cells of cloned animals should be erased in their spermatozoa or oocytes. This is supported by the fact that offspring from cloned animals do not exhibit defects at birth or during postnatal development. To test this hypothesis in cloned cattle, we compared the DNA methylation level of two imprinted genes (H19 and PEG3) and three non-imprinted genes (XIST, OCT4 and NANOG) and two repetitive elements (Satellite I and Satellite II) in blood and sperm DNAs from cloned and non-cloned bulls. We found no differences between cloned and non-cloned bulls. We also analyzed the DNA methylation levels of four repetitive elements (Satellite I, Satellite II, Alpha-satellite and Art2) in oocytes recovered from cloned and non-cloned cows. Again, no significant differences were observed between clones and non-clones. These results suggested that imprinted and non-imprinted genes and repetitive elements were properly reprogramed during gametogenesis in cloned cattle; therefore, they contributed to the soundness of cloned cattle offspring.

First cloned Bactrian camel (Camelus bactrianus) calf produced by interspecies somatic cell nuclear transfer: A step towards preserving the critically endangered wild Bactrian camels.

Studies were conducted to explore the possibility of employing dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for the development of interspecies somatic cell nuclear transfer (iSCNT) embryos using skin fibroblast cells of an adult Bactrian camel (Camelus bactrianus) and Llama (Llama glama) as donor nuclei. Also, the embryos reconstructed with Bactrian cells were transferred into the uterus of synchronized dromedary camel recipients to explore the possibility of using them as surrogate mothers. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated mature oocytes, collected from super-stimulated dromedary camels, and fused using an Eppendorf electroporator. After activation with 5μM ionomycin and 6-dimethylaminopurine, they were cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. In experiment 1, Day 7 blastocysts were stained with Hoechst to count their cell numbers, while in experiment 2, they were transferred to synchronized dromedary recipients. A lower number (P < 0.05) of blastocysts were obtained from reconstructs utilizing fibroblast cells from Llama when compared with those reconstructed with dromedary and Bactrian fibroblast cells. However, no difference was observed in their cell numbers. In experiment 2, a higher (P < 0.05) proportion of blastocysts were obtained from the cleaved embryos reconstructed with Bactrian fibroblast cells when compared to those reconstructed with dromedary cells. Twenty-six Day 7 blastocysts reconstructed with Bactrian cells were transferred to 23 synchronized dromedary recipients with 5 pregnancies established on Day 30, however, only one of the pregnancies developed to term and a healthy calf weighing 33 kgs was born after completing 392 days of gestation. Unfortunately, the calf died on day 7 due to acute septicemia. In conclusion, the present study reports, for the first time, birth of a cloned Bactrian calf by iSCNT using dromedary camel as a source for oocytes as well as a surrogate for carrying the pregnancy to term.

Aberrant expression of MICO1 and MICO1OS in deceased somatic cell nuclear transfer calves.

Incomplete reprogramming of a donor nucleus following somatic cell nuclear transfer (SCNT) results in aberrant expression of developmentally important genes, and is the primary source of the phenotypic abnormalities observed in cloned animals. Expression of non-coding RNAs in the murine Dlk1-Dio3 imprinted domain was previously shown to correlate with the pluripotency of mouse induced pluripotent stem cells. In this study, we examined the transcription of the bovine orthologs from this locus, MICO1 (Maternal intergenic circadian oscillating 1) and MICO1OS (MICO1 opposite strand), in tissues from artificially inseminated and SCNT calves that died during the perinatal period. A single-nucleotide polymorphism (SNP), a T-to-C transition, was used to analyze the allelic transcription of MICO1. Our results indicate monoallelic expression of the MICO1C allele among the six analyzed tissues (heart, liver, spleen, lung, kidney, and brain) of artificially inseminated calves, indicating that this gene locus may be imprinted in bovine. Conversely, we observed variable allelic transcription of MICO1 in SCNT calves. We asked if DNA methylation regulated the monoallelic expression of MICO1 and MICO1OS by evaluating the methylation levels of six regions within or around this locus in tissues with normal or aberrant MICO1 transcription; all of the samples from either artificially inseminated or SCNT calves exhibited hypermethylation, implying that DNA methylation may not be involved in regulating its monoallelic expression. Furthermore, three imprinted genes (GTL2, MEG9, and DIO3) nearby MICO1 showed monoallelic expression in SCNT calves with aberrant MICO1 transcription, indicating that not all of the genes in the bovine DLK1-DIO3 domain are mis-regulated.

H3K4 Methylation-Dependent Memory of Somatic Cell Identity Inhibits Reprogramming and Development of Nuclear Transfer Embryos

SummaryVertebrate eggs can induce the nuclear reprogramming of somatic cells to enable production of cloned animals. Nuclear reprogramming is relatively inefficient, and the development of the resultant embryos is frequently compromised, in part due to the inappropriate expression of genes previously active in the donor nucleus. Here, we identify H3K4 methylation as a major epigenetic roadblock that limits transcriptional reprogramming and efficient nuclear transfer (NT). Widespread expression of donor-cell-specific genes was observed in inappropriate cell types in NT embryos, limiting their developmental capacity. The expression of these genes in reprogrammed embryos arises from epigenetic memories of a previously active transcriptional state in donor cells that is characterized by high H3K4 methylation. Reducing H3K4 methylation had little effect on gene expression in donor cells, but it substantially improved transcriptional reprogramming and development of NT embryos. These results show that H3K4 methylation imposes a barrier to efficient nuclear reprogramming and suggest approaches for improving reprogramming strategies.

Dynamic Polarization and Relaxation of 75As Nuclei in Silicon at High Magnetic Field and Low Temperature

We present the results of experiments on dynamic nuclear polarization and relaxation of 75As in silicon crystals. Experiments are performed in strong magnetic fields of 4.6 T and temperatures below 1 K. At these conditions donor electron spins are fully polarized, and the allowed and forbidden electron spin resonance transitions are well resolved. We demonstrate effective nuclear polarization of 75As nuclei via the Overhauser effect on the time scale of several hundred seconds. Excitation of the forbidden transitions leads to a polarization through the solid effect. The relaxation rate of donor nuclei has strong temperature dependence characteristic of Orbach process.

Combined positive effect of oocyte extracts and brilliant cresyl blue stained recipient cytoplasts on epigenetic reprogramming and gene expression in buffalo nuclear transfer embryos.

This study examined the effects of buffalo oocyte extracts (BOE) on donor cells reprogramming and molecular characterisation of oocytes screened via brilliant cresyl blue (BCB) staining and comparison of gene expression profiles of developmentally important genes in blastocysts from IVF and cloned derived from BOE treated donor cells with BCB selected recipient cytoplasts. Relative abundance (RA) of OCT4 and NANOG was increased (P<0.05) and HDAC-1, DNMT-1, and DNMT-3A decreased (P<0.05) in extract treated cells (ETCs). This ETCs dedifferentiated into neuron-like lineage under appropriate induction condition. The RA of NASP, EEF1A1, DNMT1, ODC1 and RPS27A was increased (P<0.05) in BCB+ oocytes, whereas ATP5A1 and S100A10 increased (P<0.05) in BCB- oocytes. Total cell number and RA of OCT4, NANOG, SOX2, DNMT1, IGF2, IGF2R, MNSOD, GLUT1, BAX and BCL2 in cloned blastocysts derived from BCB+ oocytes with ETC more closely followed that of IVF counterparts compared to BCB+ oocytes with extract untreated cell and BCB- oocytes with ETC derived blastocysts. In conclusion, BOE influenced epigenetic reprogramming of buffalo fibroblasts making them suitable donors for nuclear transfer (NT). BCB staining can be effectively used for selection of developmentally competent oocytes for NT. The combined effects of epigenetic reprogramming of donor nuclei by BOE and higher nuclear reprogramming capacity of BCB+ oocytes improve developmentally important gene expression in cloned blastocysts. Whether these improvements have long-term effects on buffalo calves born following embryo transfer remains unknown.

Probability, odds and random chance: the difficult task of modulating the epigenetic profile of cloned embryos

Since the beginning of modern embryology, scientists have wondered about how a small number of totipotent embryonic cells can become an individual with a wide variety of organs and tissues with distinct functions. Also, the idea of generating a cloned animal using a nucleus from a donor cell is not recent. However, it has taken years of research to achieve this goal, especially regarding mechanisms of cell reprogramming required to return a differentiated cell to totipotency. Cloning by somatic cell nuclear transfer (SCNT) has been a valuable tool to understand epigenetic mechanisms related to cellular reprogramming. However, cloning efficiency is still low, with a low percentage of embryos resulting in healthy animals. The high attrition rate is associated with incomplete or abnormal epigenetic reprogramming, such that many cloned embryos have DNA methylation patterns different than controls, resulting in faulty gene expression and subsequent developmental failures. Attempts to improve genome reprogramming by modulation of oocyte quality and/or somatic cell plasticity, thereby increasing cloning efficiency and preventing detrimental effects on development, have proven ineffective. The recent development of DNA editing techniques may facilitate an improved understanding of cellular reprogramming and the role of DNA methylation in development. These novel tools may lead to new means to modulate epigenetic programming and inheritance, and hold great promise to assist in epigenetic remodeling of the donor nucleus. Such strategies are likely to improve the odds for successful cloning.

A Homokaryon Assay for Nucleocytoplasmic Shuttling Activity of HBV Core Protein.

Hepatitis B virus (HBV) core protein (HBc) can be present in both nucleus and cytoplasm. The arginine-rich domain (ARD) at the cytoplasmic tail of HBc contains both a nuclear localization signal (NLS) and nuclear export signal (NES). We established a homokaryon assay to detect the dynamic trafficking of HBc between nucleus and cytoplasm in hepatocytes. Using immunofluorescence assay (IFA) and PEG-induced cell-cell fusion, we demonstrated that a chimeric reporter protein of SV40 large T antigen, when fused in-frame with HBc ARD, can shuttle from a donor nuclei (green) to the recipient nuclei (red) in the context of binucleated or polynucleated hybrid cells. The shuttling activity driven by HBc ARD can be measured quantitatively by this IFA method.

33 BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED BY HAND-MADE CLONING AND IN VITRO FERTILIZATION DIFFER IN THEIR GLOBAL TRANSCRIPTOME PROFILE

Although the blastocyst rate obtained with nuclear transferred (NT) embryos is higher than that obtained following in vitro fertilization (IVF) in buffalo, the live birth rate of NT embryos is &lt;2% across different farm animal species compared with a birth rate &gt;40% obtained with IVF embryos. This is believed to be due primarily to incomplete or incorrect nuclear reprogramming of the donor somatic cell by the oocyte, which results in aberrant embryonic gene expression. We compared the global transcriptome profile of buffalo blastocysts produced by hand-made cloning (HMC) and IVF using next-generation sequencing (NGS) for discovering transcripts that are differentially expressed between the 2 types of embryos. NT blastocysts were produced using fibroblast donor cells obtained from ear skin of a buffalo bull. The semen of the same bull was used for producing genetically half-identical IVF blastocysts. Total RNA was isolated from 3 pools of Day 8 NT and IVF blastocysts, with each pool containing 40 blastocysts. Complementary DNA library was prepared and subjected to NGS on Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA). The reads generated were aligned to Bos taurus reference genome, UMD 3.1. Differential expression analysis between the 2 blastocysts types at a minimum of 2-fold change revealed that 5557 transcripts were differentially expressed, of which 584 were unique to NT blastocysts, 709 were unique to IVF blastocysts, and 4264 were expressed in both types of blastocysts. Among these transcripts, at a significance level of P &lt; 0.05, 331 transcripts were differentially expressed between the 2 blastocyst types, of which 19 were unique, 188 were down-regulated, and 143 were up-regulated in NT blastocysts. One-way ANOVA with Benjamini and Hochberg false discovery rate (FDR) correction was applied to determine the statistically significant differentially expressed transcripts. Nine of the differentially expressed transcripts (at minimal 2-fold change, P &lt; 0.05), from different functional classes (RELN, NDRG1, SULT1A1, MAP1LC3A, MTHFD1L, PCBD1, PPA2, MGST1 and PRPH) were subjected to quantitative real-time PCR analysis for validation of NGS data. Gene expression level of RELN, NDRG1, SULT1A1, MAP1LC3A, PPA2, MGST1, and PRPH was found to be up-regulated while that of MTHFD1L and PCBD1 was down-regulated (P &lt; 0.05) in NT embryos compared with IVF embryos. This pattern and the magnitude of relative gene expression level were found to be similar to that observed in NGS. These results indicate that the gene expression profile of NT embryos is very different from that of their IVF counterparts. Further analysis of these differentially expressed transcripts can help in identification of gene functional classes and pathways that are affected by the inefficient reprogramming of donor nuclei in NT embryos. Normalizing the expression of some of the differentially expressed genes may help in improving the cloning efficiency.

RE1-silencing Transcription Factor (REST) Is Required for Nuclear Reprogramming by Inhibiting Transforming Growth Factor β Signaling Pathway

Differentiated cells can be reprogrammed by transcription factors, and these factors that are responsible for successful reprogramming need to be further identified. Here, we show that the neuronal repressor RE1-silencing transcription factor (REST) is rich in porcine oocytes and requires for nuclear transfer (NT)-mediated reprogramming through inhibiting TGFβ signaling pathway. REST was dramatically degraded after oocyte activation, but the residual REST was incorporated into the transferred donor nuclei during reprogramming in NT embryos. Inhibition of REST function in oocytes compromised the development of NT embryos but not that of IVF and PA embryos. Bioinformation analysis of putative targets of REST indicated that REST might function on reprogramming in NT embryos by inhibiting TGFβ pathway. Further results showed that the developmental failure of REST-inhibited NT embryos could be rescued by treatment of SB431542, an inhibitor of TGFβ pathway. Thus, REST is a newly discovered transcription factor that is required for NT-mediated nuclear reprogramming.

Advances in equine cloning by somatic cell nuclear transfer (SCNT) technique in horses: a review

Cloning a horse means using the genetic material (DNA) from a donor horse to produce a genetically identical foal. This technique involves collecting the DNA from the donor and inserting that DNA into an egg from another mare whose, DNA content has been removed, fusing donor nucleus with enucleated recipient oocytes, which then develops as an embryo, in vitro culture of embryo and lastly transfer cultured embryo into the uterus of a recipient mare. The modification of the in vitro culture conditions which can be suitable for equine oocyte activation, oocyte maturation and embryo development are the fundamental steps for a successful in vitro procedure for somatic cell nuclear transfer (SCNT) in the horse to avoid the embryo losses. In general, few studies are available in the literature on equine in vitro embryo production and it is only recently that reports have been published on completely in vitro production of equine preimplantation embryos by means of in vitro oocyte maturation The present review discusses the latest developments in the field of equine cloning technique with the employment of SCNT. The basic understanding of SCNT for in vitro culture conditions is relevant to the increased efficiency of cloning. The available genotype can be used by SCNT which can enhance the vigour of a particular infertile or low fertile animal to produce normal fertility.

Interspecies somatic cell nuclear transfer in Asiatic cheetah using nuclei derived from post-mortem frozen tissue in absence of cryo-protectant and in vitro matured domestic cat oocytes

Recent accomplishments in the field of somatic cell nuclear transfer (SCNT) hold tremendous promise to prevent rapid loss of animal genetic resources using ex situ conservation technology. Most of SCNT studies use viable cells for nuclear transfer into recipient oocytes. However, preparation of live cells in extreme circumstances, in which post-mortem material of endangered/rare animals is improperly retained frozen, is difficult, if not impossible. This study investigated the possibility of interspecies-SCNT (iSCNT) in Asiatic cheetah (Acinonyx jubatus venaticus), a critically endangered subspecies, using nuclei derived from frozen tissue in absence of cryo-protectant at −20 °C and in vitro matured domestic cat oocytes. No cells growth was detected in primary culture of skin and tendon pieces or following culture of singled cells prepared by enzymatic digestion. Furthermore, no live cells were detected following differential viable staining and almost all cells had ruptured membrane. Therefore, direct injection of donor nuclei into enucleated cat oocytes matured in vitro was carried out for SCNT experiments. Early signs of nuclear remodeling were observed as early as 2 h post-iSCNT and significantly increased at 4 h post-iSCNT. The percentages of iSCNT reconstructs that cleaved and developed to 4–16 cell and morula stages were 32.3 ± 7.3, 18.2 ± 9.8 and 5.9 ± 4.3%, respectively. However, none of the iSCNT reconstructs developed to the blastocyst stage. When domestic cat somatic and oocytes were used for control SCNT and parthenogenetic activation, the respective percentages of oocytes that cleaved (51.3 ± 13.9 and 77.3 ± 4.0%) and further developed to the blastocyst stage (11.3 ± 3.3 and 16.8 ± 3.8%) were comparable. In summary, this study demonstrated that enucleated cat oocytes can partially remodel and reactivate non-viable nuclei of Asiatic cheetah and support its reprogramming back to the embryonic stage. To our knowledge, this is the first report of iSCNT in cheetah using non-viable frozen cells.