Efficient reverse genetics approach involving infectious subgenomic amplicon for engineering dengue virus.

Dengue virus, which belongs to the Flaviviridae family, can induce a range of symptoms from mild to severe, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. While infectious cloning technology is a useful tool for understanding viral pathogenesis and symptoms, it exhibits limitations when constructing the entire Flavivirus genome. The instability and toxicity of the genome to bacteria make its full-length construction in bacterial vectors a time-consuming and laborious process. To address these challenges, we employed the modified infectious subgenomic amplicon (ISA) method in this study, which can potentially be a superior tool for reverse genetic studies on the dengue virus. Using ISA, we generated recombinant dengue viruses de novo and validated their robust replication in both human and insect cell lines, which was comparable to that of the original strains. Moreover, the efficiency of ISA in genetically modifying the dengue virus was elucidated by successfully inserting the gene for green fluorescence protein into the genome of dengue virus serotype 4. Overall, this study highlighted the effectiveness of ISA for genetically engineering the dengue virus and provided a technical basis for a convenient reverse genetics system that could expedite investigations into the dengue virus.

Effects of klotho protein or klotho knockdown in porcine oocytes at different stages.

Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo. However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.

Porcine Follicular Fluid-Derived Exosome: The Pivotal Material for Porcine Oocyte Maturation in Lipid Antioxidant Activity.

Several studies have examined exosomes derived from porcine follicular fluid (FF), but few have reported their application in controlled experiments. The main concern in the field of embryology may be that controlled conditions, such as using a defined medium intermittently, cause poor results in mammalian oocyte maturation and embryo development. The first reason is the absence of the FF, which copes with the majority of the processes emerging in oocytes and embryos. Therefore, we added exosomes derived from porcine FF to the maturation medium of porcine oocytes. For morphological assessment, cumulus cell expansion and subsequent embryonic development were evaluated. Moreover, several stainings, such as glutathione (GSH) and reactive oxygen species (ROS), fatty acid, ATP, and mitochondrial activity, as well as evaluations of gene expression and protein analysis, were used for the functional verification of exosomes. When the oocytes were treated with exosomes, the lipid metabolism and cell survival of the oocytes were fully recovered, as well as morphological evaluations compared to the porcine FF-excluded defined medium. Therefore, controlled experiments may produce reliable data if the exosomes are treated with the desired amounts, and we suggest applying FF-derived exosomes to promote experimental data when performing controlled experiments in embryology.

MicroRNA and Messenger RNA Expression Profiles in Canine Mammary Gland Tumor.

Canine mammary gland tumor (CMT) is the most frequently diagnosed neoplasm in intact female dogs. As prognosis depends on the malignancy of tumors and metastasis levels, early and accurate diagnosis are crucial for prolongation of life expectancy. The genetic similarity of dogs with humans in addition to environmental and physiological similarities make them ideal models for the study of cancer. In this study, we analyzed differentially expressed microRNAs followed by RNA-Seq to investigate the alterations in mRNA levels based on the malignancy (benign, malignant) and the biopsy locations (tumors, surrounding normal tissues). We identified multiple breast cancer-related genes regardless of malignancy. We found cfa-miR-503 to be the only miRNA that showed altered expression in response to malignancy in CMTs. Although further validation is needed, cfa-miR-503 could be used as a potential diagnostic biomarker as well as a potential RNA-based anti-tumor drug in malignant CMTs.

Generation of a Dystrophin Mutant in Dog by Nuclear Transfer Using CRISPR/Cas9-Mediated Somatic Cells: A Preliminary Study.

Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in maintaining the genetic background using conventional breeding. In this study, we successfully generated a dystrophin mutant dog using the CRISPR/Cas9 system and somatic cell nuclear transfer. The dystrophin mutant dog displayed phenotypes such as elevated serum creatine kinase, dystrophin deficiency, skeletal muscle defects, an abnormal electrocardiogram, and avoidance of ambulation. These results indicate that donor cells with CRISPR/Cas9 for a specific gene combined with the somatic cell nuclear transfer technique can efficiently produce a dystrophin mutant dog, which will help in the successful development of gene therapy drugs for dogs and humans.

Zinc supplementation alleviates endoplasmic reticulum stress during porcine oocyte in vitro maturation by upregulating zinc transporters.

Endoplasmic reticulum (ER) stress is a major contributor to embryonic development failure. Mammalian oocytes have a high risk of exposure to cellular stress during in vitro embryo production. We investigated the effects of zinc supplementation during in vitro maturation under ER stress. We evaluated cumulus expansion, embryonic development derived by parthenogenetic activation, reactive oxygen species, protein expression of X-box binding protein 1 (XBP1), and expression of genes related to ER stress. Supplementation with 1 µg/ml zinc significantly increased the nuclear maturation of oocytes, cleavage and blastocyst formation rates, and total blastocyst cell number (p < .05). Under ER stress, zinc significantly reduced protein expression of XBP1, and increased cleavage and blastocyst rates (p < .05). Concomitantly, zinc supplementation upregulated the expression of zinc transporters (SLC39A14 and SLC39A10), PTGS2, and downregulated ER stress-related genes (sXBP1, uXBP1, ATF4, and PTPN1/PTP1B), and caspase 3. These results suggest that zinc supplementation alleviates ER stress by providing essential metal-ion transporters for oocyte maturation and subsequent embryonic development.

Failure to maintain full-term pregnancies in pig carrying klotho monoallelic knockout fetuses.

BACKGROUND: Small animals that show a deficiency in klotho exhibit extremely shortened life span with multiple aging-like phenotypes. However, limited information is available on the function of klotho in large animals such as pigs. RESULTS: In an attempt to produce klotho knockout pigs, an sgRNA specific for klotho (targeting exon 3) was designed and Cas9-sgRNA ribonucleoproteins were transfected into porcine fibroblasts. Transfected fibroblasts were cultured for one to 2 days and then directly used for nuclear transfer without selection. The cloned embryos were cultured in vitro for 7 days and analyzed to detect modifications of the klotho gene by both T7E1 and deep sequencing analysis. Modification succeeded in 13 of 20 blastocysts (65%), 8 of which (40.0%) were monoallelic modifications and 5 (25.0%) were biallelic modifications. Based on high mutation rates in blastocysts, we transferred the cloned embryos to 5 recipient pigs; 1 recipient was pregnant and 16 fetuses were recovered at Day 28 post transfer. Of the 16 fetuses, 9 were resorbing and 7 were viable. Four of 9 (44.4%) resorbing fetuses and 3 of the 7 (42.9%) viable fetuses had monoallelic modifications. Thus, 3 klotho monoallelic knockout cell lines were established by primary culture. A total of 2088 cloned embryos reconstructed with 2 frame-shifted cell lines were transferred to 11 synchronized recipients. Of the recipients, 7 of 11 eleven (63.6%) became pregnant. However, none of the pregnancies was maintained to term. To discover why klotho monoallelic knockout fetuses were aborted, expression of aging- and apoptosis-related genes and klotho protein in placentas from klotho monoallelic knockout and wild-type fetuses was investigated. Placentas from klotho monoallelic knockout fetuses showed negatively changed expression of aging- and apoptosis-related genes with lower relative expression of klotho protein. These results indicated that the reason why klotho monoallelic knockout fetuses were not maintained to term was possibly due to decreased klotho expression in placentas, negatively affecting aging- and apoptosis-related genes. CONCLUSIONS: Klotho monoallelic knockout porcine fetal fibroblasts were successfully established. However, pigs carrying klotho monoallelic knockout fetuses failed to maintain full-term pregnancy and a decrease in klotho expression in placenta likely leads to pregnancy loss.

Leptin Modulates the Metastasis of Canine Inflammatory Mammary Adenocarcinoma Cells Through Downregulation of Lysosomal Protective Protein Cathepsin A (CTSA).

Canine malignant mammary gland tumors present with a poor prognosis due to metastasis to other organs, such as lung and lymph node metastases. Unlike in human studies where obesity has been shown to increase the risk of breast cancer, this has not been well studied in veterinary science. In our preliminary study, we discovered that leptin downregulated cathepsin A, which is responsible for lysosomal-associated membrane protein 2a (LAMP2a) degradation. LAMP2a is a rate-limiting factor in chaperone-mediated autophagy and is highly active in malignant cancers. Therefore, in this study, alterations in metastatic capacity through cathepsin A by leptin, which are secreted at high levels in the blood of obese patients, were investigated. We used a canine inflammatory mammary gland adenocarcinoma (CHMp) cell line cultured with RPMI-1640 and 10% fetal bovine serum. The samples were then subjected to real-time polymerase chain reaction, Western blot, immunocytochemistry, and lysosome isolation to investigate and visualize the metastasis and chaperone-mediated autophagy-related proteins. Results showed that leptin downregulated cathepsin A expression at both transcript and protein levels, whereas LAMP2a, the rate-limiting factor of chaperone-mediated autophagy, was upregulated by inhibition of LAMP2a degradation. Furthermore, leptin promoted LAMP2a multimerization through the lysosomal mTORC2 (mTOR complex 2)/PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1)/AKT1 (Serine/threonine-protein kinase 1) pathway. These findings suggest that targeting leptin receptors can alleviate mammary gland cancer cell metastasis in dogs.

Enhancement of epigenetic reprogramming status of porcine cloned embryos with zebularine, a DNA methyltransferase inhibitor.

Aberrant epigenetic reprogramming is known to be a major cause of inefficient somatic cell nuclear transfer (SCNT) in pigs, and use of epigenetic modification agents, such as DNA methyltransferase inhibitors (DNMTis), is a promising approach for enhancing SCNT efficacy. Here, we attempted to find the optimal condition of zebularine (Zb), a DNMTi, treatment on porcine SCNT embryos during in vitro culture (IVC). As results, treatment with 5 nM Zb for 24 hr showed the highest rate of embryo development to blastocyst compared to other groups (p < .05). Also, the relative intensities of global DNA methylation levels of anti-5-methylcytosine in pseudo-pronuclear (PNC), 2-cell and 4-cell stages were significantly lower in the Zb-treated group (p < .05), however, changes in methylation levels of centromeric satellite repeat were noted only in PNC and blastocyst stages. In addition, significant positive alterations in the relative expression of genes related to pluripotency (OCT4 and SOX2), histone acetylation (HAT1, HDAC1, HDAC2, and HDAC3) and DNA methylation (DNMT1 and DNMT3a) were observed compared to the control (p < .05). In conclusion, we found that Zb could modify DNA methylation levels in the early stages of porcine SCNT embryos and promote their developmental competence.

Generation by somatic cell nuclear transfer of GGTA1 knockout pigs expressing soluble human TNFRI-Fc and human HO-1.

Herein, we successfully generated transgenic pigs expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin (HA)-tagged-human heme oxygenase-1 (hHO-1) without Gal epitope. Healthy cloned pigs were produced by somatic cell nuclear transfer (SCNT) using the genetically modified cells. The genetic disruption of the GGTA1 genes and absence of expression of BS-IB4 lectin in tail-derived fibroblast of the SCNT-generated piglets were successfully confirmed. The expression of shTNFRI-Fc and HAhHO-1 was fully identified with protective effect against oxidative stress and apoptosis stimulation. Antibody-mediated complement-dependent cytotoxicity assay for examining the immuno-reactivity of transgenically derived pig cells showed that pigs lacking GGTA1 with the expression of double genes reduce the humoral barrier to xenotransplantation, more than pigs simply expressing double genes and the wild type. Through this approach, rapid production of a pig strain deficient in various genes may be expected to be applicable for xenotransplantation research without extensive breeding protocols.

Effects of manganese on maturation of porcine oocytes in vitro and their subsequent embryo development after parthenogenetic activation and somatic cell nuclear transfer.

This study was carried out to examine the effects of manganese (Mn) on the developmental competence of porcine oocytes during in vitro maturation (IVM) after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). Upon treatment of porcine oocytes with different concentrations (0, 3, 6, and 12 ng/ml) of Mn during IVM, PA was performed to determine the optimum concentration. Following PA, the rate of blastocyst formation was higher significantly in treated porcine oocytes at 6 ng/ml of Mn than in other groups (P < 0.05). However, there was no substantial difference in the cleavage rate and total blastocyst cell numbers among all groups. SCNT was performed using the optimal concentration of Mn from PA, which showed an improved blastocyst formation rate in treated oocytes compared to that in control group (P < 0.05). However, the cleavage rate and total cell numbers per blastocyst were not different between the control and the Mn treated groups after SCNT. Additionally, oocyte nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels were assessed. There was no significant difference observed in nuclear maturation among all the groups. However, enhanced intracellular GSH levels while lower levels of ROS were seen in the Mn treated group compared to the control group (P < 0.05). Thus, these results indicate that Mn supplementation can improve the developmental competence of porcine PA and SCNT embryos by increasing GSH and decreasing ROS levels.

Improved early development of porcine cloned embryos by treatment with quisinostat, a potent histone deacetylase inhibitor.

Recently, the modification of the epigenetic status of somatic cell nuclear transfer (SCNT) embryos by treatment with histone deacetylase inhibitors (HDACis) has made it possible to alter epigenetic traits and improve the developmental competence of these embryos. In the current study, we examined the effects of an HDACi, quisinostat (JNJ), on the in vitro development of porcine cloned embryos and their epigenetic nuclear reprogramming status. SCNT embryos were cultured under various conditions, and we found that treatment with 100 nM JNJ for 24 h post activation could improve blastocyst formation rates compared to the control (P < 0.05). Therefore, this was chosen as the optimal condition and used for further investigations. To explore the effects of JNJ on the nuclear reprogramming of early stage embryos and how it improved cloning efficiency, immunofluorescence staining and quantitative real-time PCR were performed. From the pseudo-pronuclear to 2-cell stages, the levels of acetylation of histone 3 at lysine 9 (AcH3K9) and acetylation of histone 4 at lysine 12 (AcH4K12) increased, and global DNA methylation levels revealed by anti-5-methylcytosine (5-mC) antibody staining were decreased in the JNJ-treated group compared to the control (P < 0.05). However, JNJ treatment failed to alter AcH3K9, AcH4K12, or 5-mC levels at the 4-cell embryo stage. Moreover, JNJ treatment significantly upregulated the expression of the development-related genes OCT4, SOX2, and NANOG, and reduced the expression of genes related to DNA methylation (DNMT1, DNMT3a, and DNMT3b) and histone acetylation (HDAC1, HDAC2, and HDAC3). Together, these results suggest that treatment of SCNT embryos with JNJ could promote their developmental competence by altering epigenetic nuclear reprogramming events.

Sonic hedgehog signaling mediates resveratrol to improve maturation of pig oocytes in vitro and subsequent preimplantation embryo development.

The beneficial effects of resveratrol on in vitro maturation (IVM) have been explained mainly by indirect antioxidant effects and limited information is available on the underlying mechanism by which resveratrol acts directly on porcine cumulus oocyte complexes (COCs). Recently, several studies reported that sonic hedgehog (SHH) signaling mediates resveratrol to exert its biological activities. Furthermore, SHH is an important signaling molecule for follicle development, oocyte maturation, and embryo development. Therefore, to elucidate the relationship between resveratrol and SHH signaling, we designed three groups: (i) control; (ii) resveratrol; and (iii) resveratrol with cyclopamine (SHH signaling inhibitor). We evaluated the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation, expression levels of mRNAs in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Resveratrol significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), oocyte nuclear maturation, cleavage and blastocyst formation rates and total cell numbers, which were blocked in the presence of cyclopamine. At the same time, a significant increase in the expression levels of mRNAs related to cumulus expansion, oocyte maturation and SHH signaling-related mRNAs and proteins from the resveratrol treatment group was also inhibited by simultaneous addition of cyclopamine. In conclusion, our results indicate that SHH signaling mediates resveratrol to improve porcine cumulus expansion, oocyte maturation, and subsequent embryo development.

A potential role of knockout serum replacement as a porcine follicular fluid substitute for in vitro maturation: Lipid metabolism approach.

The use of supplements, such as porcine follicular fluid (pFF), fetal bovine serum and human serum albumin are widely used during in vitro maturation (IVM) in different species but these supplements contain undefined components that cause technical difficulties in standardization and influence the efficiency of IVM. Knockout serum replacement (KSR) is a synthetic protein source, without any undefined growth factors or differentiation-promoting factors. Therefore, it is feasible to use KSR as a defined component for avoiding effects of unknown molecules in an IVM system. In this study, the rates of oocyte maturation and blastocyst formation after parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) were significantly higher in the 5% KSR supplemented group than in the unsupplemented control group and more similar to those of the 10% pFF supplemented group. Moreover, the intensity of GDF9, BMP15, ROS, GSH, BODIPY-LD, BODIPY-FA, and BODIPY-ATP staining showed similar values between 5% KSR and 10% pFF, which have significant difference with control group. Most of the gene expression related to lipid metabolism with both supplements exhibited similar patterns. In conclusion, 5% KSR upregulated lipid metabolism and thereby provides an essential energy source to sustain and improve oocyte quality and subsequent embryo development after PA, SCNT, and IVF. These indications support the idea that KSR used as a defined serum supplement for oocyte IVM might be universally used in other species.

Dog cloning-no longer science fiction.

Since the generation of world's first cloned dog, Snuppy, in 2005, somatic cell nuclear transfer (SCNT) in dogs has been widely applied for producing several kinds of dogs with specific objectives. Previous studies have demonstrated that cloned dogs show normal characteristics in growth, blood parameters and behavioural aspect. Also, canine SCNT technique has been applied to propagate working dogs with excellent abilities in fields such as assistance of disabled people, drugs detection and rescue activity. Because dogs have similar habituation properties and share many characteristics including anatomic and physiological aspects with humans, they are also primary candidates for human disease models. Recently, transgenic dogs that express red fluorescent protein gene constitutively and green fluorescent protein gene conditionally have been generated. In addition, transgenic dogs with an overexpression of peroxisome proliferator-activated receptor-alpha in specific muscles were generated to enhance physical performance. In 2017, Snuppy was recloned with markedly increased pregnancy and delivery rates compared to the statistics from when Snuppy was first cloned. Such striking improvements in the cloning of dogs using SCNT procedures suggest that dog cloning could be applied in many fields of biomedical science for human diseases research, and the application of cloning is no longer science fiction.

Stimulatory Effects of Melatonin on Porcine In Vitro Maturation Are Mediated by MT2 Receptor.

Melatonin is a multifunctional molecule with numerous biological activities. The fact that melatonin modulates the functions of porcine granulosa cells via the MT2 receptor suggests the possibility of MT2 receptor-mediation for melatonin to promote cumulus expansion of porcine cumulus-oocyte complexes (COCs). Therefore, we investigated the presence of MT2 in porcine COCs, and the effects of melatonin with or without selective MT2 antagonists (luzindole and 4-P-PDOT) on this process; COCs underwent in vitro maturation culturing with six different conditions (control, melatonin, luzindole, 4-P-PDOT, melatonin + luzindole or melatonin + 4-P-PDOT). Cumulus expansion, oocyte nuclear maturation, and subsequent embryo development after parthenogenetic activation (PA) were evaluated. In experiment 1, MT2 was expressed in both oocytes and cumulus cells. In experiment 2, melatonin significantly increased the proportion of complete cumulus expansion (degree 4), which was inhibited by simultaneous addition of either luzindole or 4-P-PDOT. A similar pattern was observed in the expression of genes related to cumulus expansion, apoptosis, and MT2. In experiment 3, no significant difference was observed in immature, degenerate, and MII oocyte rates among the groups. In experiment 4, melatonin significantly increased blastocyst formation rates and total blastocyst cell numbers after PA, but these effects were abolished when either luzindole or 4-P-PDOT was added concomitantly. In conclusion, our results indicate that the MT2 receptor mediated the stimulatory effects of melatonin on porcine cumulus expansion and subsequent embryo development.

The HDAC Inhibitor LAQ824 Enhances Epigenetic Reprogramming and In Vitro Development of Porcine SCNT Embryos.

BACKGROUND/AIMS: Hypoacetylation caused by aberrant epigenetic nuclear reprogramming results in low efficiency of mammalian somatic cell nuclear transfer (SCNT). Many epigenetic remodeling drugs have been used in attempts to improve in vitro development of porcine SCNT embryos. In this study, we examined the effects of LAQ824, a structurally novel histone acetylase inhibitor, on the nuclear reprogramming and in vitro development of porcine SCNT embryos. METHODS: LAQ824 treatment was supplemented during the culture of SCNT embryos. The reprogramming levels were measured by immunofluorescence and quantified by image J software. Relative expression levels of 18 genes were analyzed by quantitative real-time PCR. RESULTS: 100 nM LAQ824 treatment of post-activation SCNT embryos for 24 h significantly improved the subsequent blastocyst formation rate. The LAQ824 treatment enhanced histone 3 lysine 9 (H3K9) levels, histone 4 lysine 12 (H4K12) levels, and reduced global DNA methylation levels as well as anti-5-methylcytosine (5-mC) at the pseudo-pronuclear and 2-cell stages. Furthermore, LAQ824 treatment positively regulated the mRNA expression of genes for histone acetylation (HAT1, HDAC1, 2, 3, and 6), DNA methylation (DNMT1, 3a and 3b), development (Pou5f1, Nanog, Sox2, and GLUT1) and apoptosis (Bax, Bcl2, Caspase 3 and Bak) in blastocysts. CONCLUSION: Optimum exposure (100 nM for 24 h) to LAQ824 post-activation improved the in vitro development of porcine SCNT embryos by enhancing levels of H3K9 and H4K12, reducing 5-mC, and regulating gene expression.

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.

Melatonin regulates lipid metabolism in porcine oocytes.

It is being increasingly recognized that the processes of lipogenesis and lipolysis are important for providing an essential energy source during oocyte maturation and embryo development. Recent studies demonstrated that melatonin has a role in lipid metabolism regulation, including lipogenesis, lipolysis, and mitochondrial biogenesis. In this study, we attempted to investigate the effects of melatonin on lipid metabolism during porcine oocyte in vitro maturation. Melatonin treatment significantly enhanced the number of lipid droplets (LDs) and upregulated gene expression related to lipogenesis (ACACA, FASN, PPARγ, and SREBF1). Oocytes treated with melatonin formed smaller LDs and abundantly expressed several genes associated with lipolysis, including ATGL, CGI-58, HSL, and PLIN2. Moreover, melatonin significantly increased the content of fatty acids, mitochondria, and ATP, as indicated by fluorescent staining. Concomitantly, melatonin treatment upregulated gene expression related to fatty acid ß-oxidation (CPT1a, CPT1b, CPT2, and ACADS) and mitochondrial biogenesis (PGC-1α, TFAM, and PRDX2). Overall, melatonin treatment not only altered both the morphology and amount of LDs, but also increased the content of fatty acids, mitochondria, and ATP. In addition, melatonin upregulated mRNA expression levels of lipogenesis, lipolysis, ß-oxidation, and mitochondrial biogenesis-related genes in porcine oocytes. These results indicated that melatonin promoted lipid metabolism and thereby provided an essential energy source for oocyte maturation and subsequent embryonic development.

Melatonin influences the sonic hedgehog signaling pathway in porcine cumulus oocyte complexes.

Melatonin, which is synthesized in the pineal gland and peripheral reproductive organs, has antioxidant properties and regulates physiological processes. It is well known that melatonin affects in vitro maturation (IVM) of oocytes and embryonic development in many species. However, beneficial effects of melatonin on IVM have been explained mainly by indirect antioxidant effects and little information is available on the underlying mechanism by which melatonin directly acts on porcine cumulus oocyte complexes (COCs). Sonic hedgehog (Shh) signaling is important for follicle development, oocyte maturation, and embryo development, and there may be a relationship between melatonin and Shh signaling. To examine this, we designed three groups: (i) control, (ii) melatonin (10-9  mol/L), and (iii) melatonin with cyclopamine (2 µmol/L; Shh signaling inhibitor). The aim of this study was to investigate the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation (PA), gene expression in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Melatonin significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), PA blastocyst formation rates, and total cell numbers, which were inhibited by addition of cyclopamine. Simultaneously, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, and Has2) and Shh signaling-related genes (Shh, Pthc1, Smo, and Gli1) and proteins (Ptch1, Smo, and Gli1) in cumulus cells was upregulated in the melatonin-treated group, and these effects were also inhibited by cyclopamine. In conclusion, our results suggest that Shh signaling mediates effects of melatonin to improve porcine cumulus expansion and subsequent embryo development.