Somatic Cell Nucleus Transfer Research Articles

Omne vivum ex ovo: the oocyte reprogramming and remodeling activities.

Understanding the establishment of post-fertilization totipotency has broad implications for modern biotechnologies. This review summarizes the current knowledge of putative egg components governing this process following natural fertilization and after somatic cell nuclear transfer. The mammalian oocyte is a unique cell, and comprehending its physiology and biology is essential for understanding fertilization, totipotency and early events of embryogenesis. Consequently, research in these areas influences the outcomes of various technologies, for example, the production and conservation of laboratory and large animals with rare and valuable genotypes, the rescue of the species near extinction, as well as success in human assisted reproduction. Nevertheless, even the most advanced and sophisticated reproductive technologies of today do not always guarantee a favorable outcome. Elucidating the interactions of oocyte components with its natural partner cell - the sperm or an 'unnatural' somatic nucleus, when the somatic cell nucleus transfer is used is essential for understanding how totipotency is established and thus defining the requirements for normal development. One of the crucial aspects is the stoichiometry of different reprogramming and remodeling factors present in the oocyte and their balance. Here, we discuss how these factors, in combination, may lead to the formation of a new organism. We focus on the laboratory mouse and its genetic models, as this species has been instrumental in shaping our understanding of early post-fertilization events.

Nucleus reprogramming/remodeling through selective enucleation (SE) of immature oocytes and zygotes: a nucleolus point of view.

It is now approximately 25 years since the sheep Dolly, the first cloned mammal where the somatic cell nucleus from an adult donor was used for transfer, was born. So far, somatic cell nucleus transfer, where G1-phase nuclei are transferred into cytoplasts obtained by enucleation of mature metaphase II (MII) oocytes followed by the activation of the reconstructed cells, is the most efficient approach to reprogram/remodel the differentiated nucleus. In general, in an enucleated oocyte (cytoplast), the nuclear envelope (NE, membrane) of an injected somatic cell nucleus breaks down and chromosomes condense. This condensation phase is followed, after subsequent activation, by chromatin decondensation and formation of a pseudo-pronucleus (i) whose morphology should resemble the natural postfertilization pronuclei (PNs). Thus, the volume of the transferred nuclei increases considerably by incorporating the content released from the germinal vesicles (GVs). In parallel, the transferred nucleus genes must be reset and function similarly as the relevant genes in normal embryo reprogramming. This, among others, covers the relevant epigenetic modifications and the appropriate organization of chromatin in pseudo-pronuclei. While reprogramming in SCNT is often discussed, the remodeling of transferred nuclei is much less studied, particularly in the context of the developmental potential of SCNT embryos. It is now evident that correct reprogramming mirrors appropriate remodeling. At the same time, it is widely accepted that the process of rebuilding the nucleus following SCNT is instrumental to the overall success of this procedure. Thus, in our contribution, we will mostly focus on the remodeling of transferred nuclei. In particular, we discuss the oocyte organelles that are essential for the development of SCNT embryos.

The expression, function, and utilization of Protamine1: a literature review.

ObjectiveProtamine 1 (PRM1) is specific in sperm and plays essential roles in fertilization, also a member of cancer testis antigen (CTA) family. This study aims to summarize the expression and function of PRM1 in spermatogenesis, and to broaden the current knowledge and inspire future development of PRM1-based therapeutic strategies in cancer treatment and nanomedicine.BackgroundThe protamine proteins, are characterized by an arginine-rich core and cysteine residues. Humans express two types of protamine: PRM1 and PRM2. The abnormal expression or proportion of PRM1 and PRM2 is known to be associated with subfertility and infertility, especially for PRM1 which is highly evolutionary conserved in mammalians and expressed in all vertebrates. Biological functions of PRM1 have been unveiled in diverse cellular processes, such as tumorigenesis, somatic cell nucleus transfer, and drug delivery systems. Moreover, PRM1 is identified as a CTA in chronic leukemia (CLL) and colorectal cancer (CRC).MethodsLiterature was obtained using PubMed and the keywords protamine 1, PRM1, or P1, from January 1, 1980, through July 20, 2021. We also collect the additional evidence through screening references of articles identified through the PubMed searches.ConclusionsPRM1 is well-studied in male infertility, and further researches and attempts to develop PRM1 as novel tumor marker, as well as drug delivery vector, will be of important clinical significance.

208 Effect of growth factors and reprogramming molecules on induction to multipotency of dermal fibroblasts from colocolo (Leopardus colocolo)

Reprogramming of terminally differentiated cells to higher plasticity levels can be achieved with small molecules. This can be of value for somatic cell nucleus transfer, deriving multi and pluripotent cells and conservation purposes. Recently, induced mesenchymal stem cells were derived from differentiated human and mouse cells by using small molecules and growth factors. The pampas cat or colocolo (Leopardus colocolo) is a South American felid categorrized as near threatened by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Major historical threats to the pampas cat include illegal hunting, habitat loss or transformation, and conflict retaliation for poultry predation. Here, we tested 5-azacytidine (an epigenetic modifier) and A8301 (a potent inhibitor of transforming growth factor-β type I receptor superfamily), linked to platelet-rich plasma (PRP) and platelet-derived growth factor (PDGF-B) to induce changes in the expression of pluripotency genes and differentiation capacity of colocolo fibroblasts towards other mesodermal lineages. For this, dermal fibroblasts were treated with (I) 5-azacytidine + PRP + A8301 + VitC, or (II) 5- azacytidine + VitC + A8301 + PDFG for 12 days. On Days 0, 5, and 12 of reprogramming, expression of OCT4, NANOG, E-cadherin and SNAIL was evaluated by reverse transcription-PCR, and tri-lineage differentiation was induced. For treatment I, no statistical difference was found in the expression of OCT4 and NANOG. Chondrogenic and osteogenic differentiation was observed. In treatment II, significant expression of OCT4 and NANOG (P<0.05) was induced, and reprogrammed fibroblasts were differentiated into chondrogenic and osteogenic lineages. Immunohistochemistry positivity for OCT4 was detected in treatment II. In summary, we showed that dermal fibroblasts of pampas cat can be reprogrammed into cells with multipotent characteristics, particularly when a cocktail of 5-azacytidine + VitC + A8301 + PDFG was used. Treatment I probably failed because of other growth factors and proteins present in PRP, which might inhibit successful reprogramming or activate other pathways leading to a nonmultipotent phenotype. Further refinements of these protocols are required to improve the reprogramming protocol. This in turn should help us obtain cells that can be used in nucleus transfer or cellular therapies in endangered felid species.

In vitro production of canine blastocysts

Though blastocyst production in vitro has been successful in several animal species, a culture system to produce viable and normal canine blastocysts in vitro remains to be established. In this study, we report the development of an in vitro culture system for canine preimplantation embryos produced via parthenogenetic activation (PA) and somatic cell nucleus transfer (SCNT). Our results show that the medium developed by us, named “Qingdao Agricultural University's (QAU)-4 medium”, successfully breaks the developmental arrest observed at the eight-cell stage in canine embryos grown in other culture systems. The blastocysts produced in QAU-4 displayed normal blastocyst structures, including a clear inner cell mass and blastocyst cavity. We also found that blastocyst formation in PA embryos cultured in QAU-4 medium was quite high, though this was not so in the case of SCNT embryos. However, supplementation of QAU-4 medium with 100 nM of scriptaid caused a sharp increase in blastocyst formation in SCNT embryos. After culture, hatched blastocysts were also observed to successfully adhere to collagen-coated dishes, where further growth and differentiation occurred. To our knowledge, this is the first in vitro canine preimplantation embryo culture system that can successfully produce canine blastocysts.

Atypical chemokine receptors 1, 2, 3 and 4: Expression and regulation in the endometrium during the estrous cycle and pregnancy and with somatic cell nucleus transfer–cloned embryos in pigs

Atypical chemokine receptor (ACKR) 1, ACKR2, ACKR3, and ACKR4, chemokine decoy receptors that lack G-protein-mediated signaling pathways, internalize and degrade chemokines to control their availability and function. Chemokines play important roles in the endometrium during the estrous cycle and pregnancy, but the expression and regulation of ACKRs have not been determined in pigs. Therefore, we examined the expression of ACKRs in the endometrium throughout the estrous cycle and pregnancy and in conceptus tissues in pigs. ACKR1, ACKR2, ACKR3, and ACKR4 mRNA was expressed in the endometrium, with higher levels of ACKR3 on day 12 of the estrous cycle than in pregnancy and higher levels of ACKR4 on day 15 of pregnancy than in the estrous cycle. ACKR1, ACKR2, and ACKR3, but not ACKR4, mRNA was detected in conceptus and chorioallantoic tissues during pregnancy. ACKR2 and ACKR3 mRNA and ACKR4 protein were mainly localized to luminal epithelial cells and weakly to glandular epithelial cells in the endometrium. Increasing doses of progesterone increased the expression of ACKR2 and ACKR4 and decreased the expression of ACKR3 in endometrial tissues. On day 12 of pregnancy, the expression of ACKR4 mRNA was lower in the endometria of gilts with somatic cell nucleus transfer–derived conceptuses than in the endometria of gilts carrying conceptuses derived from natural mating. These results indicate that the expression of ACKRs is dynamically regulated at the maternal–conceptus interface, suggesting that ACKR proteins might play critical roles in regulating endometrial chemokines to support the establishment and maintenance of pregnancy in pigs.

Cloning of a gene-edited macaque monkey by somatic cell nuclear transfer.

Cloning of macaque monkeys by somatic cell nucleus transfer (SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey (Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing (see accompanying paper). Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood mRNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.

Nucleus transfer efficiency of ear fibroblast cells isolated from Bama miniature pigs at various ages.

Somatic cell nucleus transfer (SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer (NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts (P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells (85% to 91%) than those at 6 and 12 months (66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group (8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

Colcemid treatment during oocyte maturation improves preimplantation development of cloned pig embryos by influencing meiotic progression and cytoplasmic maturation.

The objective of this study was to examine the effects of colcemid treatment during oocyte in vitro maturation (IVM) and embryonic development after parthenogenetic activation (PA) and somatic-cell nucleus transfer (SCNT) in pigs. Immature oocytes were treated with colcemid from 0 to 22, 38 to 42, or 0 to 22 hr followed by 38 to 42 hr during IVM (designated as COL0-22, COL38-42, and COL0-22/38-42, respectively). The proportion of oocytes reaching the germinal vesicle (GV)/GV breakdown (GVBD) stage after 22 hr of IVM was higher in COL0-22 (98.4%) than in controls not exposed to colcemid (68.7%). The proportion of metaphase-II (MII) oocytes after 30 hr of IVM was higher in control (79.6%) than in COL0-22 oocytes (61.7%); overall nuclear progression to the MII stage was not influenced by colcemid treatment by the end of the IVM period (93.8, 86.7, 86.8, and 84.8% for control, COL0-22, COL38-42, and COL0-22/38-42, respectively). COL0-22 oocytes showed higher intra-oocyte glutathione content (1.7 vs. 1.0-1.3 pixels/oocyte) and increased blastocyst formation after PA (68.7% vs. 42.5-52.2%) and SCNT (39.4% vs. 16.3-28.6%) than control, COL38-42, and COL0-22/38-42 oocytes. Colcemid treatment for 0-22 and 0-22/38-42 hr of IVM also stimulated the expression of cyclin-dependent kinase 1 (CDK1), proliferating cell nuclear antigen (PCNA), and extracellular signal-regulated kinase 2 (ERK2) mRNAs. Our results thus demonstrate that the presence of colcemid during the early stage of IVM stimulates preimplantation development of PA and SCNT porcine embryos by improving the cytoplasmic microenvironment.

Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells.

The transfer of somatic cell nuclei into oocytes can give rise to pluripotent stem cells that are consistently equivalent to embryonic stem cells, holding promise for autologous cell replacement therapy. Although methods to induce pluripotent stem cells from somatic cells by transcription factors are widely used in basic research, numerous differences between induced pluripotent stem cells and embryonic stem cells have been reported, potentially affecting their clinical use. Because of the therapeutic potential of diploid embryonic stem-cell lines derived from adult cells of diseased human subjects, we have systematically investigated the parameters affecting efficiency of blastocyst development and stem-cell derivation. Here we show that improvements to the oocyte activation protocol, including the use of both kinase and translation inhibitors, and cell culture in the presence of histone deacetylase inhibitors, promote development to the blastocyst stage. Developmental efficiency varied between oocyte donors, and was inversely related to the number of days of hormonal stimulation required for oocyte maturation, whereas the daily dose of gonadotropin or the total number of metaphase II oocytes retrieved did not affect developmental outcome. Because the use of concentrated Sendai virus for cell fusion induced an increase in intracellular calcium concentration, causing premature oocyte activation, we used diluted Sendai virus in calcium-free medium. Using this modified nuclear transfer protocol, we derived diploid pluripotent stem-cell lines from somatic cells of a newborn and, for the first time, an adult, a female with type 1 diabetes.

Pluripotent-related gene expression analyses in single porcine recloned embryo

The developmental ability among embryos produced by three different techniques were examined: there were no significant differences in the developmental rate in porcine embryos produced by in vitro fertilization (IVF) and first generation of somatic cell nucleus transfer (SCNT), but the developmental rate dropped sharply at the 2- to four-cell stage in recloned (second generation of SCNT) embryos. In most recloned embryos, Oct4 and Klf4 were under-expressed at all stages, whereas Sox2 and Nanog were over-expressed at the two-cell stage. In contrast, Nanog was absent in IVF and SCNT embryos at the two-cell stage. The recloned embryos were treated with valproic acid to enhance developmental capacity and this led to an increase in the rate of blastocyst formation and total cell number compared with the findings for untreated recloned embryos (29.8 vs. 12.4%, 39 vs. 25, respectively, p<0.05).

Intracytoplasmic sperm injection (ICSI)-mediated transgenesis in mice.

Over the years many well-described techniques for the introduction of transgene DNA into host organisms have been used, including pronuclear injection, in vitro fertilization-mediated transgenesis, transfection of ES and spermatogenic cells, nuclear transfer of somatic cell nuclei, and lentiviral vectors. The application of these techniques has been limited however either by the time and effort to be executed or by their narrow efficiency with large transgenes. The greatest advantage of intracytoplasmic sperm injection (ICSI)-mediated transgenesis is precisely its ability to stably introduce large DNA molecules into the genome of host organisms with relatively high efficiency, as compared to alternative procedures. In mice, this procedure has been shown to be a reproducible method to generate transgenic offspring with a high efficiency. Recently, it proved also to be a viable method to generate transgenic rats and pigs, and as such, it is foreseen with great interest for the production of transgenic farm animals, where it would constitute an important tool for the production of recombinant proteins and livestock improvement.

Treatment of donor cells with trichostatin A improves in vitro development and reprogramming of buffalo (Bubalus bubalis) nucleus transfer embryos

It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 μM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4–0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos.

The ups and downs of somatic cell nucleus transfer (SCNT) in humans

Achieving successful somatic cell nuclear transfer (SCNT) in the human and subhuman primate relative to other mammals has been questioned for a variety of technical and logistical issues. Here we summarize the gradual evolution of SCNT technology from the perspective of oocyte quality and cell cycle status that has recently led to the demonstration of feasibility in the human for deriving chromosomally normal stem cells lines. With these advances in hand, prospects for therapeutic cloning must be entertained in a conscientious, rigorous, and timely fashion before broad spectrum clinical applications are undertaken.

Improving development of cloned goat embryos by supplementing α-lipoic acid to oocyte in vitro maturation medium

α-Lipoic acid (LA) is a powerful antioxidant for clinical therapy of some metabolic diseases, but there are few reports about the effect of LA on animal occyte in vitro maturation (IVM). The objective of this study is to investigate the effect of supplementing LA to IVM medium on subsequently developmental competence of goat cloning embryos after somatic cell nucleus transfer (SCNT). Twenty-five micromolars LA significantly increased 12% oocyte maturation rate from control 57.8% to treated group 69.8% (P < 0.05). The reconstructed rate of cloning embryos in LA supplement group (67.3%) was significantly higher than control (56.5%, P < 0.05). Although the SCNT embryo cleavage rates did not have significant difference between the two groups (42.0% vs. 47.9%, P > 0.05), LA supplement group had significantly higher blastocyst formation rate and hatched rate than control (24.0% vs. 18.4% and 37.0% vs. 30.9%, respectively, P < 0.05). In addition, supplementing LA significantly reduced the cellular apoptosis rate of nucleus transfer blastocysts by inhibiting the expression of apoptotic activators, such as Bax, Bad, Caspase-3, and CytC genes and promoting cumulus-oocyte complexes to synthesize glutathione (GSH) and express antioxidant enzymes such as GPX4 and SOD genes. In conclusion, supplement of LA to oocyte IVM medium could improve the maturation rate and antioxidant ability of oocytes and increase the developmental competence of oocytes after SCNT.

Immunobiology of embryonic stem cells: Foreign mtDNA as an immunological barrier in SCNT derived embryonic stem cells transplantation

Objectives: Using somatic cell nucleus transfer (SCNT) for embryonic stem cell (ESC) extraction, the nucleus of a somatic cell is transferred to the cytoplasm of an enucleated egg. ESCs created from the inner cell mass of the blastocyst are not genetically identical with the nucleus donor. The mitochondrial DNA (mtDNA) resides inside the cytoplasm of the egg. This study aimed to investigate the immunological response against mtDNA in ESC transplantation.

49 EFFECTS OF CULTURE CONDITIONS AND GENE TRANSFECTION ON THE DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Somatic cell nucleus transfer (SCNT) and in vitro culture of reconstructed embryos are the pivotal steps for successful cloning and generation of transgenic cattle. The aim of the study was to determine the influence of different cell fusion parameters, maturation, and culture conditions and the type of a cell line (bovine fetal fibroblast cell lines with or without gene transfection) on SCNT blastocyst development. Slaughterhouse-derived oocytes were matured for 17 h in TCM-199 (Sigma, St. Louis, MO, USA) supplemented with 0.05 µg mL–1 of epidermal growth factor (EGF) and 15 IU mL–1 of hCG/eCG (Intervet, PG600) or 10 µg mL–1 of FSH and 12.5 mU mL–1 of LH (Sioux Biochemical Inc., Sioux Center, IA, USA). Four fetal fibroblast cell lines (4 to 5 passages) and identical cell lines transfected with plasmid containing either human erythropoietin, FSH, growth hormone, or insulin-coding cDNA under β-casein promoter (7 to 9 passages) were used for SCNT. Cell fusion was induced by 2 direct-current pulses in 0.5 or 0.2 micro fusion chambers (Eppendorf Multiporator) using one of the following treatments: 100V for 15 µs (F1), 65V for 25 µs (F2), 65V for 20 µs (F3; all in a 0.5-mm chamber), or 36V for 25 µs (F4; 0.2-mm chamber). Fused complexes were activated with 4 µg mL–1 of Ca-ionophore for 4 min and then incubated for 5 h in 2 mM DMAP. The embryos were cultured in SOFaaci medium (Holm et al. 1999) or in commercial SOF medium (Minitüb GmbH, Tiefenbach, Germany) for 7 days. Data were analysed by ANOVA and the chi-square test. The results of the study showed that the cleavage rate of the reconstructed embryos was influenced by the fusion regimen (P &lt; 0.05) but not by the donor cell type (P &lt; 0.05). Treatments F2 and F3 resulted in cleavage rates higher (P &lt; 0.05) than F1 and F4 (77.2, 82.0, 62.8, and 63.1%, respectively). Blastocyst yield was not significantly influenced by the different in vitro maturation (IVM) media – altogether, addition of FSH/LH resulted in 14.6% (158/1079) and EGF + hCG/eCG in 13.2% (73/554) of blastocysts (P &lt; 0.05). The combination of TCM-199 + FSH/LH and SOFaaci resulted in 19.6% (79/403) blastocysts compared with 12.4% (74/596) when the same IVM medium and commercial SOF were used (P &lt; 0.05). The use of transgenic cell lines for cloning led to a lower overall blastocyst rate (10.9%, 38/348) than use of non-transfected cell lines (17.7%, 115/651; P &lt; 0.05), whereas the differences were 5.6 and 4.1 percentage points for SOF and SOFaaci, respectively. There were no significant differences between the individual cell lines within a cell line type. In conclusion, the optimization of the fusion parameters and in vitro culture (IVC) conditions led to improved blastocyst yields. In vivo development potential of the generated embryos still has to be evaluated in further studies. This study was supported by Project EU29023 of Enterprise Estonia, CCRMB, targeted grant SF1080045s07, and grant P8001 from the Estonian University of Life Sciences.

Immunobiology of SCNT Derived Embryonic Stem Cells: Pivotal Role of mtDNA in Rejection of Transplanted Stem Cells

Background: For regenerative therapies, somatic cell nucleus transfer (SCNT) can be used to transfer the nucleus of a somatic cell into the cytoplasm of an enucleated egg. The resulting SCNT-embryo has the capability to develop to the blastocyst stage. Subsequently, embryonic stem cells (ESCs) can be derived from the inner cell mass of these blastocysts. These ESCs are genetically identical with the donor nucleus, with the exception of mitochondrial DNA (mtDNA) from the receiving egg. This study aimed to investigate the role of mtDNA in ESC transplantation and its immunological recognition. Methods: SCNT-ESCs were generated by transferring donor cells from Balb/C background into enucleated oocytes from BDF1 background. 1×106 of these SCNT-ESCs or SCNT-ESC-derived cardiomyocytes (CMs) were injected into Balb/C (mitochondria-incompatible) or BDF1 (nucleus-incompatible) mice (n=10). After 5 days, cellular and humoral responses were investigated by ELISPOT, donorspecific antibodies, histopathology, and confocal immunofluorescence microscopy. Immunodeficient SCIDbeige mice were used as controls (n=6). In vivo bioluminescence imaging was used to visualize cell survival longitudinal. The amount of mitochondria in undifferentiated ESCs was compared to ESC-derived CMs using the TMRM assay (Figure 1).[Figure 1]Results: Using ELISPOT assays for Th1, Th2, and Th17-cells, we found that foreign mitochondria caused a similar immune response compared to ESCs with foreign nuclear DNA, both being significant higher than in SCIDbeige (p< 0.001). Similar results were observed for the humoral response. Re-injection of ESCs resulted in an adaptive immune response, highlighting the role of T-cell mediated rejection as shown by ELISPOT assays and in vivo bioluminescence imaging. Antigenicity of ESCs increased with their differentiation into CMs, correlating with increased MHCI-expression and amount of mitochondrial antigens (Figure 1). Conclusion: mtDNA is coding for 13 proteins, which surprisingly resulted in an adaptive immune response and donorspecific antibody production after transplantation. Upon ESC differentiation into CMs we observed an increase of mitochondrial antigens resulting in a greater immunological recognition of transplanted cells. In summary, SCNT-derivates are not sufficient immune privileged and undergo immune rejection after transplantation.

330 Regenerative Therapy after Myocardial Infarction: Role of mtDNA in SCNT Derived Embryonic Stem Cells

For regenerative therapies, somatic cell nucleus transfer (SCNT) can be used to transfer the nucleus of a somatic cell into the cytoplasm of an enucleated egg. Subsequently, embryonic stem cells (ESCs) can be derived from the inner cell mass of these blastocysts. These ESCs are genetically identical with the donor nucleus, with the exception of mitochondrial DNA (mtDNA) from the receiving egg. This study aimed to investigate the role of mtDNA in ESC transplantation and its immunological recognition.

Silencing myostatin gene by RNAi in sheep embryos

Myostatin (MSTN) gene is described as a negative regulator of the skeletal muscle growth. Controlling MSTN gene expression by genetic manipulation could accelerate the muscle growth and meat production of livestock animals. In the present study, several siRNAs targeting sheep MSTN gene were designed and their interfering efficiency was evaluated in vitro. The present study showed that one of the siRNAs, PSL1, could down-regulate the expression of MSTN significantly. PSL1 was ligated into lentivirus vector, GP-Supersilencing, to construct a siRNA expression lentivirus vector. Fibroblast cells were infected by lentivirus particles and positive cells were isolated by flow cytometry. Nucleus of the positive cell was transferred into enucleated oocytes of sheep. The present study showed that 99.4% of the sorted cells displayed green fluorescence. After enucleation of oocytes with microinjection, about 20% of reconstructed embryos can be developed into morulas, and strong green fluorescence could be observed using a fluorescence microscope. This method can be available to produce transgenic cell line for somatic cell nucleus transfer for transgenic animals.