Nuclear Transfer Research Articles

The Effects of an In Vitro Oocyte Maturation System and Chlorogenic Acid Supplementation during Embryo Culture on the Development of Porcine Cloned Embryos Derived from Native Vietnamese Ban Pigs

The aim of this study was to improve the production efficiency of Vietnamese native Ban pig embryos using somatic cell nuclear transfer (SCNT). Fibroblast cells from Ban pigs were injected into the enucleated cytoplasts of crossbred gilts, and the reconstructed embryos were subsequently cultured. In the first experiment, cytoplasts were isolated from oocytes matured in either a defined porcine oocyte medium (POM) or in TCM199 medium supplemented with porcine follicular fluid. Both media were supplemented with gonadotropic hormones, either for the first 22 h of in vitro maturation (IVM) or for the entire 44 h of IVM. In the second experiment, the reconstructed SCNT embryos were cultured with or without 50 μM chlorogenic acid (CGA). Furthermore, this study examined parthenogenetic embryos. The IVM medium and duration of hormone treatment did not affect embryo development. CGA supplementation to the culture medium significantly increased blastocyst formation rates in parthenogenetic embryos but not in SCNT embryos. However, CGA supplementation significantly reduced the apoptotic index in blastocysts regardless of embryo source. In conclusion, the IVM method did not affect SCNT embryo production, while CGA supplementation during embryo culture improved the quality of SCNT embryos in indigenous pig breeds.

Cytokine supplemented maturation medium improved development to term following somatic cell nuclear transfer (SCNT) in cattle.

In vitro maturation is an important process in the production of embryos. It has been shown that three cytokines, fibroblast growth factor 2, leukemia inhibitory factor and insulin-like growth factor 1 (FLI), increased efficiency of in vitro maturation, somatic cell nuclear transfer (SCNT) blastocyst production, and in vivo development of genetically engineered piglets. Assess effects of FLI on oocyte maturation, quality of oocytes, and embryo development in bovine in vitro fertilisation (IVF) and SCNT. Cytokine supplementation resulted in significant increases in maturation rates and decreased levels of reactive oxygen species. Oocytes matured in FLI had increased blastocyst rates when used in IVF (35.6%vs 27.3%, P <0.05) and SCNT (40.6%vs 25.7%, P <0.05). SCNT blastocysts contained significantly more inner cell mass and trophectodermal cells when compared to the control group. Importantly, SCNT embryos derived from oocytes matured in FLI medium resulted in a four-fold increase in full-term development compared to control medium (23.3%vs 5.3%, P <0.05). Relative mRNA expression analysis of 37 genes associated with embryonic and fetal development revealed one gene had differential transcript abundance in metaphase II oocytes, nine genes at the 8-cell stage, 10 genes at the blastocyst stage in IVF embryos and four genes at the blastocyst stage in SCNT embryos. Cytokine supplementation increased efficiency of in vitro production of IVF and SCNT embryos and in vivo development of SCNT embryos to term. Cytokine supplementation is beneficial to embryo culture systems, which may shed light on requirements of early embryo development.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.

ZFP57 regulates DNA methylation of imprinted genes to facilitate embryonic development of somatic cell nuclear transfer embryos in Holstein cows.

Aberrant epigenetic nuclear reprogramming, especially imprinting pattern disorders, is one of the major causes of failure of clone development from somatic cell nuclear transfer (SCNT). Previous studies showed that ZFP57 is a key protein required for imprint maintenance after fertilization. In this study, we found that imprinting control regions in several imprinted genes were significantly hypomethylated in cloned embryos compared with in vitro fertilization embryos, indicating a loss of imprinted gene methylation. The ZFP57 expression was capable of maintaining the correct degree of methylation at several imprinting control regions and correcting abnormal hypomethylation. Moreover, we successfully obtained bovine fetal fibroblasts overexpressing ZFP57, which were used as donors for SCNT. Our results demonstrated that overexpression of ZFP57 increased total and trophectoderm cell numbers and the ratio of inner cell mass to total cells, reduced the apoptosis rate and significantly enhanced the development of SCNT blastocysts in vitro, ultimately achieving a degree of methylation similar to that in in vitro fertilization embryos. We concluded that overexpression of ZFP57 in donor cells provided an effective method for enhancing nuclear reprogramming and developmental potential in SCNT embryos. The ZFP57 protein played a key role in maintaining the methylation of imprinted genes during early embryonic development, which may be effective for enhanced SCNT in cattle.

Live and carcass production traits for progeny of purebred sires in comparison with the clone of a USDA prime yield grade one carcass.

Cloning is a technology by which an animal's tissue can be salvaged and replicated. Carcasses that grade USDA prime-yield grade 1 (P1) represent a rare and antagonistic outcome and are a goal for terminal sire selection in the United States. A terminal sire progeny test generated offspring for a crossbred bull (14% Zebu, 86% Angus; ALPHA), born in 2012 via somatic cell nuclear transfer (SCNT) from a carcass that graded P1. ALPHA progeny (steers and heifers) were compared against progeny of three purebred (Angus; Charolais; Simmental) reference sires. Live production traits included weaning weight, morbidity, mortality, and days on feed; carcass traits included abscessed liver frequency and lung lesion frequency, individual quality and yield grade (YG) parameters, and carcass value. Observed carcass traits for progeny from the Angus, Charolais, and Simmental sires were reflective of the carcass outcomes expected for each sire's respective breed. Calves sired by the Angus were the earliest maturing indicated by the youngest chronological age at harvest (P ≤ 0.02) concomitant with the most backfat (P < 0.01), and the greatest marbling scores (P < 0.01). Calves sired by the Charolais had the heaviest carcass weight (P = 0.04), greatest cutability as assessed by USDA calculated YG (P < 0.01) and were the heaviest muscled based on "longissimus" muscle area (P < 0.01). ALPHA-sired calves were the most similar in carcass outcomes to calves sired by the Simmental, combining advantageous quality and yield parameters to produce an intermediate for carcass quality and yield. The economic value of moderate carcass outcomes is reflected in the carcass value per century weight, in which ALPHA-sired steers tended (P = 0.07) to be of the greatest value compared to other sire groups. ALPHA progeny performed comparably to high-performing reference sires for terminal sire production traits and the P1 genetics in which ALPHA was cloned have economical and biological value in modern U.S. beef production.

Haploidy of somatic cells in mouse oocyte using somatic cell nuclear transfer

Haploidy of somatic cells in mouse oocyte using somatic cell nuclear transfer

In vitro genome editing activity of Cas9 in somatic cells after random and transposon-based genomic Cas9 integration.

Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo. Direct in vivo edits may facilitate the production of genetically engineered pigs by integrating Cas9 into the porcine genome. Cas9 expressing cells were generated by either random integration or transposon-based integration of Cas9 and used as donor cells in SCNT. In total, 15 animals were generated that carried a transposon-based Cas9 integration and two pigs a randomly integrated Cas9. Cas9 expression was confirmed in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver in two boars. Overall, Cas9 expression was higher for transposon-based integration, except in tonsils and liver. To verify Cas9 activity, fibroblasts were subjected to in vitro genome editing. Isolated fibroblasts were transfected with guide RNAs (gRNA) targeting different genes (GGTA1, B4GALNT2, B2M) relevant to xenotransplantation. Next generation sequencing revealed that the editing efficiencies varied (2-60%) between the different target genes. These results show that the integrated Cas9 remained functional, and that Cas9 expressing pigs may be used to induce desired genomic modifications to model human diseases or further evaluate in vivo gene therapy approaches.

Parahydrogen-Induced Hyperpolarization of Unsaturated Phosphoric Acid Derivatives

Parahydrogen-induced nuclear polarization offers a significant increase in the sensitivity of NMR spectroscopy to create new probes for medical diagnostics by magnetic resonance imaging. As precursors of the biocompatible hyperpolarized probes, unsaturated derivatives of phosphoric acid, propargyl and allyl phosphates, are proposed. The polarization transfer to 1H and 31P nuclei of the products of their hydrogenation by parahydrogen under the ALTADENA and PASADENA conditions, and by the PH-ECHO-INEPT+ pulse sequence of NMR spectroscopy, resulted in a very high signal amplification, which is among the largest for parahydrogen-induced nuclear polarization transfer to the 31P nucleus.

SIRT7 affects autophagy and activation of hepatic stellate cells by regulating the acetylation level of high mobility group protein 1

ObjectivePreventing the progression of hepatic fibrosis is an important strategy to improve the prognosis of liver disease. The purpose of this study was to investigate the role of sirtuin7 (SIRT7) and high mobility group box 1 (HMGB1) acetylation in the occurrence and development of hepatic fibrosis. Materials and methodsHepatic fibrosis mice model was induced by CCl4. TGF-β1 was used to activated quiescent hepatic stellate cell (qHSC) into activated HSC (aHSC). Hematoxylin-eosin evaluated hepatic fibrosis in vivo, and the distribution of α-smooth muscle actin (α-SMA) or HMGB1 was detected by immunohistochemistry or immunofluorescence. The expressions of SIRT7, autophagy related proteins, and HSC activation-related proteins were detected by Western blot. Immunoprecipitation detected the acetylation level of HMGB1. Lysine mutants of HMGB1 were constructed in vitro to explore the acetylation sites of HMGB1. ResultsHepatocyte autophagy and activation levels were enhanced in CCl4 group or aHSC group, and the acetylation level of HMGB1 was increased. Nuclear transfer of HMGB1 occurred in aHSC, and HMGB1was mainly distributed in cytoplasm. The expression of SIRT7 in CCl4 group or aHSC group was most significantly decreased, and knockdown of SIRT7 leads to increased levels of HSCs autophagy and activation. Overexpression of SIRT7 or interference of HMGB1 alone in aHSC can reduce the level of autophagy and activation of aHSC. However, continued overexpression of SIRT7 in shHMGB1-aHSC could not reduce the autophagy and activation levels of aHSC. Among the 11 Flag-HMGB1 mutants, the acetylation level of K86R-Flag-HMGB1 was the lowest. The acetylation level of K86R-Flag-HMGB1 did not change due to SIRT7 downregulation. ConclusionThis study proved that SIRT7 can directly target the K86R site of HMGB1 and participate in regulating the expression and distribution of HMGB1, thus affecting the autophagy and activation level of HSCs.

Interspecific Nuclear Transfer Blastocysts Reconstructed from Arabian Oryx Somatic Cells and Domestic Cow Ooplasm.

Cloning, commonly referred to as somatic cell nuclear transfer (SCNT), is the technique of enucleating an oocyte and injecting a somatic cell into it. This study was carried out with interspecific SCNT technology to clone the Arabian Oryx utilizing the oryx's fibroblast cells and transfer it to the enucleated oocytes of a domestic cow. The recipient oocytes were extracted from the cows that had been butchered. Oryx somatic nuclei were introduced into cow oocytes to produce embryonic cells. The study was conducted on three groups, Oryx interspecific somatic cell nuclear transfer into enucleated oocytes of domestic cows, cow SCNT "the same bovine family species", used as a control group, and in vitro fertilized (IVF) cows to verify all media used in this work. The rates of different embryo developmental stages varied slightly (from 1- cell to morula stage). Additionally, the oryx interspecies Somatic cell nuclear transfer blastocyst developmental rate (9.23%) was comparable to that of cow SCNT (8.33%). While the blastula stage rate of the (IVF) cow embryos exhibited a higher cleavage rate (42%) in the embryo development stage. The results of this study enhanced domestic cow oocytes' ability to support interspecific SCNT cloned oryx, and generate a viable embryo that can advance to the blastula stage.

Current Status of Renal Xenotransplantation and Next Steps.

Renal transplantation is the preferred treatment of ESKD, but the shortage of suitable donor kidneys from the cadaver pool means that many patients with ESKD will not receive a kidney transplant. Xenotransplantation has long represented a solution to the kidney shortage, but the occurrence of antibody-mediated rejection has precluded its clinical development. Developments in somatic cell nuclear transfer in pigs and gene editing tools have led to the creation of new donor pigs with greatly improved crossmatches to patients. In addition, improvements in preclinical kidney xenotransplant survival using new anti-CD40/CD154-based immunosuppression have pushed xenotransplantation to the point where it is reasonable to consider initiating a clinical trial to evaluate this potential therapy in patients.

REVIEW ON TRANSGENIC TECHNOLOGY IN LIVESTOCK: CURRENT STATUS AND FUTURE HORIZONS

&#x0D; &#x0D; &#x0D; Transgenic technologies are one of the most promising arenas in the field of biotechnology. These techniques employ genetic engineering techniques to insert foreign genes of interest into genome of an individual. These foreign genes are inherited and expressed by the recipient‟s offspring.&#x0D; &#x0D; &#x0D; &#x0D; Two things determine the success of any transgenic technique namely the transport of foreign piece of DNA across the recipient‟s cell membrane and delivery of that DNA across the nuclear membrane gaining access to the chromosomes of the recipient. Many transgenic techniques are employed for genetic manipulation like microinjection, somatic cell nuclear transfer, chemical methods, restriction enzyme mediated gene transfer, retrovirus mediated gene transfer as well as embryonic stem cell and sperm mediated DNA transfer etc. The transgenic animals can be employed in drug discovery, disease research and xenotransplantation etc. This review summarizes various transgenic techniques, applications and future prospects of the transgenic technology.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D;

Blastocyst complementation and interspecies chimeras in gene edited pigs.

The only curative therapy for many endstage diseases is allograft organ transplantation. Due to the limited supply of donor organs, relatively few patients are recipients of a transplanted organ. Therefore, new strategies are warranted to address this unmet need. Using gene editing technologies, somatic cell nuclear transfer and human induced pluripotent stem cell technologies, interspecies chimeric organs have been pursued with promising results. In this review, we highlight the overall technical strategy, the successful early results and the hurdles that need to be addressed in order for these approaches to produce a successful organ that could be transplanted in patients with endstage diseases.

18 Hybrid lamb of domestic sheep and argali produced by somatic cell nuclear transfer

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

19 Exploring the expression of protamine 1 as a “xeroprotectant” in somatic cell nuclear transfer using sheep freeze-dried adult fibroblast

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Emerging technologies and ethics—exogenic chimeric humanized organs

Organ transplantation is limited due to the scarcity of donor organs. In order to expand the supply of organs for transplantation, interspecies chimeras have been examined as a potential future source of humanized organs. Recent studies using gene editing technologies in combination with somatic cell nuclear transfer technology and hiPSCs successfully engineered humanized skeletal muscle in the porcine embryo. As these technologies progress, there are ethical issues that warrant consideration and dialogue.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned, ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

BackgroundSCNT (somatic cell nuclear transfer) is of great significance to biological research and also to the livestock breeding. However, the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods. This indicates the potential epigenetic variations between them. DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences. In this study, ASMT (acetylserotonin-O-methyltransferase) ovarian overexpression transgenic goat was produced by using SCNT. To investigate whether there are epigenetic differences between cloned and WT (wild type) goats, WGBS (whole-genome bisulfite sequencing) was used to measure the whole-genome methylation of these animals.ResultsIt is observed that the different mCpG sites are mainly present in the intergenic and intronic regions between cloned and WT animals, and their CG-type methylation sites are strongly correlated. DMR (differentially methylated region) lengths are located around 1000 bp, mainly distributed in the exonic, intergenic and intronic functional domains. A total of 56 and 36 DMGs (differentially methylated genes) were identified by GO and KEGG databases, respectively. Functional annotation showed that DMGs were enriched in biological-process, cellular-component, molecular-function and other signaling pathways. A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.ConclusionThe differences in methylation genes among the tested animals have been identified. A total of 10 DMGs associated with growth and development were identified between cloned and WT animals. The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT. These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology, particularly in goats.

Profiling the transcriptomic signatures and identifying the patterns of zygotic genome activation – a comparative analysis between early porcine embryos and their counterparts in other three mammalian species

BackgroundThe transcriptional changes around zygotic genome activation (ZGA) in preimplantation embryos are critical for studying mechanisms of embryonic developmental arrest and searching for key transcription factors. However, studies on the transcription profile of porcine ZGA are limited.ResultsIn this study, we performed RNA sequencing in porcine in vivo developed (IVV) and somatic cell nuclear transfer (SCNT) embryo at different stages and compared the transcriptional activity of porcine embryos with mouse, bovine and human embryos. The results showed that the transcriptome map of the early porcine embryos was significantly changed at the 4-cell stage, and 5821 differentially expressed genes (DEGs) in SCNT embryos failed to be reprogrammed or activated during ZGA, which mainly enrichment to metabolic pathways. c-MYC was identified as the highest expressed transcription factor during ZGA. By treating with 10,058-F4, an inhibitor of c-MYC, the cleavage rate (38.33 ± 3.4%) and blastocyst rate (23.33 ± 4.3%) of porcine embryos were significantly lower than those of the control group (50.82 ± 2.7% and 34.43 ± 1.9%). Cross-species analysis of transcriptome during ZGA showed that pigs and bovines had the highest similarity coefficient in biological processes. KEGG pathway analysis indicated that there were 10 co-shared pathways in the four species.ConclusionsOur results reveal that embryos with impaired developmental competence may be arrested at an early stage of development. c-MYC helps promote ZGA and preimplantation embryonic development in pigs. Pigs and bovines have the highest coefficient of similarity in biological processes during ZGA. This study provides an important reference for further studying the reprogramming regulatory mechanism of porcine embryos during ZGA.

Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

CNS Delivery of Nucleic Acid Therapeutics: Beyond the Blood-Brain Barrier and Towards Specific Cellular Targeting.

Nucleic acid-based therapeutic molecules including small interfering RNA (siRNA), microRNA(miRNA), antisense oligonucleotides (ASOs), messenger RNA (mRNA), and DNA-based gene therapy have tremendous potential for treating diseases in the central nervous system (CNS). However, achieving clinically meaningful delivery to the brain and particularly to target cells and sub-cellular compartments is typically very challenging. Mediating cell-specific delivery in the CNS would be a crucial advance that mitigates off-target effects and toxicities. In this review, we describe these challenges and provide contemporary evidence of advances in cellular and sub-cellular delivery using a variety of delivery mechanisms and alternative routes of administration, including the nose-to-brain approach. Strategies to achieve subcellular localization, endosomal escape, cytosolic bioavailability, and nuclear transfer are also discussed. Ultimately, there are still many challenges to translating these experimental strategies into effective and clinically viable approaches for treating patients.