Embryo Cloning Research Articles

Legality extent of therapeutic cloning in Islamic Fiqh and comparative laws—A comparative analytical study

The rapid advancement of biotechnologies involves human subjects that brings forth intricate ethical, legal, social, and religious challenges. Recent breakthroughs in cloning and stem cell research have generated new hopes as well as presents considerable promise for treating severe diseases and regenerative medicine. In this regard, for advancements in biotechnology to be clinically applicable in medicine, it’s essential to comprehend both scientific principles and ethical implications, taken into consideration the scientific roadblocks impeding advancement in therapeutic cloning such as tumorigenicity, epigenetic reprogramming and interspecies pathogen transfer. As research in this area progresses, it will clarify that stem cells can be obtained from various sources, including therapeutic cloning, which involves cloning embryos from the nuclei of somatic cells or cloning individual organs in the laboratory. This article studies the legitimacy extent of therapeutic cloning in Islamic Fiqh and comparative law, using the comparative analytical approach. Thus, we have reached a number of results, the most important of which is that the Islamic Fiqh perspective on the illegality of extracting stem cells by creating therapeutic embryos using Somatic Cell Nuclear Transfer (SCNT). It is deemed unacceptable to create an embryo for the purpose of its development and then destroying it for the purpose of stem cells, as this is unacceptable that turns the human being into a source for spare parts. Conversely, cloning specific organs and tissues in the laboratory for medical purposes is considered permissible, provided that it does not harm anyone or violate their sanctity that is consistent with many comparative laws.

Optical tweezer-assisted cell pairing and fusion for somatic cell nuclear transfer within an open microchannel.

Somatic cell nuclear transfer (SCNT), referred to as somatic cell cloning, is a pivotal biotechnological technique utilized across various applications. Although robotic SCNT is currently available, the subsequent oocyte electrical activation/reconstructed embryo electrofusion is still manually completed by skilled operators, presenting challenges in efficient manipulation due to the uncontrollable positioning of the reconstructed embryo. This study introduces a robotic SCNT-electrofusion system to enable high-precision batch SCNT cloning. The proposed system integrates optical tweezers and microfluidic technologies. An optical tweezer is employed to facilitate somatic cells in precisely reaching the fusion site, and a specific polydimethylsiloxane (PDMS) chip is designed to assist in positioning and pairing oocytes and somatic cells. Enhancement in the electric field distribution between two parallel electrodes by PDMS pillars significantly reduces the required external voltage for electrofusion/electrical activation. We employed porcine oocytes and porcine fetal fibroblasts for SCNT experiments. The experimental results show that 90.56% of oocytes successfully paired with somatic cells to form reconstructed embryos, 76.43% of the reconstructed embryos successfully fused, and 70.55% of these embryos underwent cleavage. It demonstrates that the present system achieves the robotic implementation of oocyte electrical activation/reconstructed embryo electrofusion. By leveraging the advantages of batch operations using microfluidics, it proposes an innovative robotic cloning procedure that scales embryo cloning.

Developmental Competence of Somatic Cell Nuclear Transfer Embryos and Interspecies ICSI Zygotes From Bovine Small Antral Follicles.

Assisted reproductive technologies (ART) play a crucial role in conserving threatened wildlife species such as Bos gaurus. ART requires a large number of mature oocytes, and small antral follicles (SAFs) in the ovary are often used to obtain abundant sources of bovine oocytes. However, oocytes from SAFs often experience difficulty completing maturation and obtaining high quality and quantity of blastocyst formation compared to fully grown oocytes. This study aimed to increase the number of high-quality mature oocytes and improve their potential for ART applications in cloned and interspecies intracytoplasmic sperm injection (ICSI) embryos by utilising L-ascorbic acid (LAA) in pre invitro maturation (pre-IVM) culture. First, oocytes isolated from SAFs were cultured with the duration of pre-IVM 0, 6, 8, 10 h and different concentrations of LAA to determine good conditions for oocyte maturation. Then, mature oocytes were assessed for their developmental competence through parthenogenesis, cloned and interspecies ICSI embryos. The results showed that 8-h pre-IVM with 50 μg/mL LAA improved the maturation rate and developmental competence of parthenogenetic and clone embryos, especially, improving the high blastocyst quality by increasing cell number and expression of histone acetylation at lysine 9 (H3K9ac). In addition, the culture process improved the nuclear reprogramming of somatic cells after nuclear transfer into mature oocytes, resulting in an increased hatching rate of cloned embryos. It also enhanced the activation and the pronuclear formation rate of Gaurus-Taurus zygotes. Overall, the established pre-IVM culture method enhanced the meiotic and developmental competence of embryos. This procedure opened hope for the preservation of endangered species and other applications.

Cellular mechanisms of monozygotic twinning: clues from assisted reproduction.

Monozygotic (MZ) twins are believed to arise from the fission of a single fertilized embryo at different stages. Monochorionic MZ twins, who share one chorion, originate from the splitting of the inner cell mass (ICM) within a single blastocyst. In the classic model for dichorionic MZ twins, the embryo splits before compaction, developing into two blastocysts. However, there are a growing number of ART cases where a single blastocyst transfer results in dichorionic MZ twins, indicating that embryo splitting may occur even after blastocyst formation. For monochorionic MZ twins, we conducted a comprehensive analysis of the cellular mechanisms involved in ICM splitting, drawing from both ART cases and animal experiments. In addition, we critically re-examine the classic early splitting model for dichorionic MZ twins. We explore cellular mechanisms leading to two separated blastocysts in ART, potentially causing dichorionic MZ twins. Relevant studies including research articles, reviews, and conference papers were searched in the PubMed database. Cases of MZ twins from IVF clinics were found by using combinations of terms including 'monozygotic twins' with 'IVF case report', 'ART', 'single embryo transfer', or 'dichorionic'. The papers retrieved were categorized based on the implicated mechanisms or as those with unexplained mechanisms. Animal experiments relating to MZ twins were found using 'mouse embryo monozygotic twins', 'mouse 8-shaped hatching', 'zebrafish janus mutant', and 'nine-banded armadillo embryo', along with literature collected through day-to-day reading. The search was limited to articles in English, with no restrictions on publication date or species. For monochorionic MZ twins, ART cases and mouse experiments demonstrate evidence that a looser ICM in blastocysts has an increased chance of ICM separation. Physical forces facilitated by blastocoel formation or 8-shaped hatching are exerted on the ICM, resulting in monochorionic MZ twins. For dichorionic MZ twins, the classic model resembles artificial cloning of mouse embryos in vitro, requiring strictly controlled splitting forces, re-joining prevention, and proper aggregation, which allows the formation of two separate human blastocysts under physiological circumstances. In contrast, ART procedures involving the transfer of a single blastocysts after atypical hatching or vitrified-warmed cycles might lead to blastocyst separation. Differences in morphology, molecular mechanisms, and timing across various animal model systems for MZ twinning can impede this research field. As discussed in future directions, recent developments of innovative in vitro models of human embryos may offer promising avenues for providing fundamental novel insights into the cellular mechanisms of MZ twinning during human embryogenesis. Twin pregnancies pose high risks to both the fetuses and the mother. While single embryo transfer is commonly employed to prevent dizygotic twin pregnancies in ART, it cannot prevent the occurrence of MZ twins. Drawing from our understanding of the cellular mechanisms underlying monochorionic and dichorionic MZ twinning, along with insights into the genetic mechanisms, could enable improved prediction, prevention, and even intervention strategies during ART procedures. N/A.

Experiences with transvaginal Ovum Pick-Up (OPU) in sows

Transvaginal ultrasound-guided Ovum Pick-Up (OPU) is an established technique in other species. Due to several challenges, there are few publications addressing the procedure in sows. An efficient OPU technique may allow for the collection of numerous oocytes from valuable sows for porcine in vitro embryo production, gene editing and cloning programmes, or cryopreservation. We aimed to improve transvaginal OPU and equipment for this technique in sows. In experiment 1, we conducted 13 OPU sessions on three Landrace x Large White hybrid sows under general anaesthesia, while the second experiment explored OPU in non-sedated animals (N = 6) physically restrained in a commercial claw trimming chute. The experiments resulted in 6.6 ± 5.6 (mean ± SD) and 7.7 ± 8.9 recovered cumulus-oocyte complexes per session, respectively. Post-mortem examination of the pelvic and abdominal cavities of the three sows subjected to repeated OPU sessions did not reveal major acute or chronic pathological lesions. The only sow which was inseminated after the experiment delivered 16 liveborn piglets at term. Salivary cortisol levels increased during the procedure in non-sedated and physically restrained sows but returned to baseline 1 h later (n = 5), indicating a short-term stress response. The described OPU technique and equipment have the potential to retrieve considerable numbers of oocytes by repeated procedures on valuable mature sows. Follow-up studies are needed to optimize the efficiency of the aspiration of high-quality oocytes and to describe the developmental competence of these OPU-derived oocytes. It is also essential to further investigate sow welfare during and after the procedure before recommending porcine transvaginal OPU as a sustainable and welfare-friendly procedure.

Review: Reproductive physiology of bison and application of assisted reproductive technologies to their conservation.

Bison are an ecologically and culturally important species on the European and North American continents. Their near extirpation was met with conservation efforts that prevented their extinction but left few animals or highly fragmented populations. Contemporary conservation efforts are focused on building ecologically and genetically sustainable bison herds for long-term conservation of the species. Assisted reproductive technologies (ARTs) can play a key role in building these herds by facilitating the movement of genetics in the form of gametes and embryos, while protecting animal well-being and ensuring biosecurity of existing bison herds. In addition, ARTs such as gamete and embryo cryopreservation can be used to protect against future losses of genetic diversity through biobanking. In this review, a brief summary of basic bison reproductive physiology is presented followed by an overview of the current state of ART in Bison bison (American bison) and Bison bonasus (European bison or wisent). Research on ART ranging from artificial insemination to in vitro embryo transfer and cloning is discussed with particular regard to the application of ART for conservation purposes. While significant progress has been made in ART for bison, there are still many opportunities to improve these technologies and expand their impact for bison conservation.

Genetic Variation among Somatic Embryo Clones of Nordmann Fir Grown as Christmas Trees

Somatic embryogenesis (SE) cloning techniques for Nordmann fir Christmas tree production have been pursued and refined in Denmark, and three SE clonal trials with 201 SE clones from seeds of 27 Nordmann fir trees and seedlings from bulk harvest seeds in Georgia, Caucasus, were established from 2014 to 2015. This study was the first to evaluate the genetic performance of these SE clones in different sites. In addition, the performance of SE clones and (zygotic) normal seedlings in the field was compared in this study. This study was based on three key groups of traits for Christmas tree production, measured 5 and 6 years after planting: growth, commercial tree quality, and disorder. Seedlings grew faster and had better Christmas tree quality than SE clones. There were significant family effects for all traits, indicating that a good family also produced good clones on average. Growth and disorder traits were under moderate genetic control, with estimated broad-sense heritability (H2) from 0.19 to 0.31 and from 0.20 to 0.28, respectively. Quality traits had low to moderate H2, ranging from 0.09 to 0.24. Significant genotype-by-environment interactions were shown for Christmas tree quality, post-harvest needle retention, and branch angle, suggesting the importance of choosing the right clone at each site. There was no significant interaction in height among the years. Superior clones, compared to the unbred seed source, were identified by combining several important traits. Overall, the somatic clones were well established but had slower growth compared to standard seedlings.

Double cytoplast embryonic cloning improves in vitro but not in vivo development from mitotic pluripotent cells in cattle.

Cloning multiple animals from genomically selected donor embryos is inefficient but would accelerate genetic gain in dairy cattle breeding. To improve embryo cloning efficiency, we explored the idea that epigenetic reprogramming improves when donor cells are in mitosis. We derived primary cultures from bovine inner cell mass (ICM) cells of in vitro fertilized (IVF) embryos. Cells were grown feeder-free in a chemically defined medium with increased double kinase inhibition (2i+). Adding recombinant bovine interleukin 6 to 2i+ medium improved plating efficiency, outgrowth expansion, and expression of pluripotency-associated epiblast marker genes (NANOG, FGF4, SOX2, and DPPA3). For genotype multiplication by embryonic cell transfer (ECT) cloning, primary colonies were treated with nocodazole, and single mitotic donors were harvested by mechanical shake-off. Immunofluorescence against phosphorylated histone 3 (P-H3) showed 37% of nocodazole-treated cells in metaphase compared to 6% in DMSO controls (P < 1 × 10−5), with an average of 53% of P-H3-positive cells expressing the pluripotency marker SOX2. We optimized several parameters (fusion buffer, pronase treatment, and activation timing) for ECT with mitotic embryonic donors. Sequential double cytoplast ECT, whereby another cytoplast was fused to the first cloned reconstruct, doubled cloned blastocyst development and improved morphological embryo quality. However, in situ karyotyping revealed that over 90% of mitotic ECT-derived blastocysts were tetraploid or aneuploid with extra chromosomes, compared to less than 2% in the original ICM donor cells. Following the transfer of single vs. double cytoplast embryos, there was no difference between the two methods in pregnancy establishment at D35 (1/22 = 5% vs. 4/53 = 8% for single vs. double ECT, respectively). Overall, post-implantation development was drastically reduced from embryonic mitotic clones when compared to somatic interphase clones and IVF controls. We conclude that mitotic donors cause ploidy errors during in vitro development that cannot be rescued by enhanced epigenetic reprogramming through double cytoplast cloning.

Sperm-borne proteins improve rabbit cloning efficiency via regulating embryonic cleavage and epigenetics.

Somatic cell nuclear transfer (SCNT) shows great application value in the generation of transgenic animals, protection of endangered species, and therapeutic cloning. However, the cloning efficiency is still very low, which greatly restricts its application. Compared to fertilized embryos, cloned embryos lack the sperm proteins, which are considered to play an important role in embryonic development. Here, we compared the sperm proteome, with that of donor fibroblasts and oocytes, and identified 342 proteins unique to sperm, with 42 being highly expressed. The 384 proteins were mainly enriched in the categories of post-translational modification and cytoskeletal arrangement. Extracts of soluble sperm or fibroblast proteins were injected into cloned embryos, and the result showed that injection of sperm protein significantly inhibited abnormal embryonic cleavage, significantly decreased the level of trimethylated histone H3 Lys9 (H3K9me3) and the apoptotic index, and increased the inner cell mass (ICM)-to-trophectoderm (TE) ratio. More importantly, the sperm proteins also significantly enhanced the birthrate. The results of in vitro and in vivo experiments demonstrate that sperm-derived proteins improve embryo cloning efficiency. Our findings not only provide new insights into ways to overcome low cloning efficiency, but also add to the understanding of sperm protein function.

Chromatin openness of donor cells is directly correlated with the in vitro developmental capabilities of cloned buffalo embryos.

The Switch/sucrose nonfermentable (SWI/SNF) chromatin remodelling complex is closely related to chromatin openness and gene transcriptional activity. To understand if the chromatin openness of donor cells was related to the development efficiency of somatic cell cloning embryos, two buffalo fetal fibroblasts (BFF), BFF1 and BFF3, with significantly different cloned blastocyst development rates (18.4% and 30.9% respectively), were selected in this study. The expression of SWI/SNF complex genes, chromatin openness, and transcript level of these two cell lines were analysed, and the effect of ATP on the expression of the SWI/SNF complex genes was further explored. The results showed that compared with BFF1, the expression of SWI/SNF complex family genes was higher in BFF3 at the G0/G1 phase, where SMARCC1, SMARCC2 and SMARCE1 were significantly different (p < .05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results showed that, at the genome-wide level, BFF3 had more open chromatin, especially which having more open chromatin peaks at SMARCA4, SMARCA2, and RBPMS2 (RNA Binding Protein, mRNA Processing Factor 2) sites. In total, 2,712 differentially expressed genes (DEGs) were identified by the RNA-Seq method, with 1380 up- and 1332 down-regulated genes in BFF3. Interestingly, the ATPase-related genes ATP1B1 and ATP11A were extreme significantly up-regulated in BFF3 (p < .01). The ATP content and the expression of SWI/SNF complex genes in both BFF1 and BFF3 decreased when treated with rotenone. The above results demonstrated that the SWI/SNF complex contributed to chromatin opening, and chromatin opening of donor cells was essential for cloned embryo development.

Individual variation in buffalo somatic cell cloning efficiency is related to glycolytic metabolism.

Mammalian individuals differ in their somatic cell cloning efficiency, but the mechanisms leading to this variation is poorly understood. Here we found that high cloning efficiency buffalo fetal fibroblasts (BFFs) displayed robust energy metabolism, looser chromatin structure, high H3K9 acetylation and low heterochromatin protein 1α (HP1α) expression. High cloning efficiency BFFs had more H3K9ac regions near to the upstream of glycolysis genes by ChIP-seq, and involved more openness loci related to glycolysis genes through ATAC-seq. The expression of these glycolysis genes was also found to be higher in high cloning efficiency BFFs by qRT-PCR. Two key enzymes of glycolysis, PDKs and LDH, were confirmed to be associated with histone acetylation and chromatin openness of BFFs. Treatment of low cloning efficiency BFFs with PS48 (activator of PDK1) resulted in an increase in the intracellular lactate production and H3K9 acetylation, decrease in histone deacetylase activity and HP1α expression, less condensed chromatin structure and more cloning embryos developing to blastocysts. These results indicate that the cloning efficiency of buffalo somatic cells is associated with their glycolytic metabolism and chromatin structure, and can be improved by increasing glycolytic metabolism.

Expression analysis of OsSERK, OsLEC1 and OsWOX4 genes in rice (Oryza sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.

Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.

‘The machine runs itself’: law is technology and Australian embryo and human cloning law

ABSTRACT Technology law scholarship has a tendency towards the dramatic. Technology causes disruption. Law must catch-up; it must ensure potential benefits from technology and avoid potential harms. There are even concerns that law, as an organiser of human life, is itself becoming eclipsed by forms of technological management. What is often not focused on is the practical process through which concerns about technology become transmuted into legal forms within specific jurisdictions. This paper examines the 23 years of Australian law concerning embryos and human cloning. Inspired by Carl Schmitt’s criticism of modernity’s political institutions and the laws they produce, what is identified is a machine that runs itself. It is shown to be a highly automated process whereby technical experts manage competing values. Rather than law regulating technology or technology regulating law; the Australian study suggests that law and its making, is technological.

Application of the modified handmade cloning technique to pigs.

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

Robotic Label-Free Precise Oocyte Enucleation for Improving Developmental Competence of Cloned Embryos.

The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. Our method is expected to improve the extremely low success rate of animal cloning in the future.

Manipulation of gametes and embryosin the pig

Summary. Several manipulation techniques including nuclear injections, nuclear transplantation, embryo splitting, chimaera production and sperm injection are discussed with special reference to their application in the pig. The nuclear injection technique is likely to be of greatest use for gene transfer. Gene transfer is feasible for pig embryos. but it is very inefficient. Efficiencies of various steps in the successful production of transgenic offspring as summarized from several published references are as follows: immediate survival of embryos following injection (60%). offspring produced from injected embryos (15%), offspring with an integrated foreign gene (24%) and offspring expressing the integrated foreign gene (60%). The overall efficiency is about 1%. Potential uses for nuclear transplantation are for the production of gynogenetic and androgenetic embryos and for cloning embryos The feasibility of producing androgenetic and gynogenctic embryos will depend on whether in the pig. as in the mouse, the paternal and maternal genomes function differently. Thc feasibility of cloning by nuclear transplantation will depend upon the timing of developmental events in pig embryos and the development of methods for reprogramming nuclei. Pig embryos have been cloned by embryo splitting. Chimaera production and fertilization by sperm injection are likely to be feasible for pig embryos but have not yet found application.

Porcine somatic cell nuclear transfer using telomerase reverse transcriptase-transfected mesenchymal stem cells reduces apoptosis induced by replicative senescence

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

The moral and legal foundations of bioethics in the context of human rights: legal theory and international practice

Modern processes of globalization taking place in the field of law are a great challenge to the idea of human nature, which is recognized in Ukraine as the highest social value, as well as to the concept and essence of law itself. In our opinion, this is a threat on a global scale and necessitates the search for an adequate response to the threat from the scientific and technical process in the field of biomedicine, both for the natural (physical) existence of man and the preservation of his moral identity. In fact, these foundations have become the prerequisites for the development of the science of bioethics. Bioethics studies controversial and ambiguous issues and proposes a humanitarian examination, which aims to assess the arguments in favor of the development of human creativity, health and prevention of premature death, and arguments in favor of preserving human identity in its spiritual and physical integrity. The purpose of the article is to study the essence of controversial bioethical problems, the reasons for their occurrence and prospects for solving these problems. human, manipulation of stem cells and others. Bioethical issues usually include the ethical issues of abortion; contraception and new reproductive technologies (artificial insemination, surrogacy); conducting experiments on humans and animals; obtaining informed consent and ensuring patients' rights; determination of death, suicide and euthanasia; problems in relation to dying patients (hospices); demographic policy and family planning; genetics (including problems of genome research, genetic engineering and gene therapy); transplantology; health equity; human cloning, manipulation of stem cells and others. These issues related to the progress of genetics, genomics, pharmacology, transplantation, biotechnology, cloning are becoming increasingly important as a direction of international law in the context of ensuring and protecting human rights. IN legal literature indicates the formation of "biolaw", "bioethical legislation", "bioethical human rights". Thus there is a combination of possibilities and purposes of medicine and law. In our article, we have explored only some of these issues, which are currently the most relevant, debatable, and therefore require detailed analysis. These include, in our view, the legal status of the embryo, therapeutic and reproductive cloning, abortion, the use of assisted reproductive technologies and organ transplantation. In order to adequately cover these issues, we compare the rules of law governing these debatable issues with the views of church representatives and scholars on these issues. We also proposed changes that need to be made to the legislation of Ukraine so that the rules of law governing these issues meet the moral and ethical principles. As a conclusion is marked, that as bioethics as science dealing with survival combines in itself biological knowledge and general human values, then it is possible to consider natural human rights, her honour and dignity morally-legal principles of bioethics, a self right and law must become on defence of that, in particular, with the aim of providing of natural (physical) existence of man, and maintenance of her moral identity. Keywords: human rights, moral, bioethics, abortion, reproductive technologies, cloning.

Maternal H3K27me3-dependent autosomal and X chromosome imprinting.

Genomic imprinting and X-chromosome inactivation (XCI) are classic epigenetic phenomena that involve transcriptional silencing of one parental allele. Germline-derived differential DNA methylation is the best-studied epigenetic mark that initiates imprinting, but evidence indicates that other mechanisms exist. Recent studies have revealed that maternal trimethylation of H3 on lysine 27 (H3K27me3) mediates autosomal maternal allele-specific genesilencing and has an important role in imprinted XCI through repression of maternal Xist. Furthermore, loss of H3K27me3-mediated imprinting contributes to the developmental defects observed in cloned embryos. This novel maternal H3K27me3-mediated non-canonical imprinting mechanism further emphasizes the important role of parental chromatin in development and could provide the basis for improving the efficiency of embryo cloning.