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Background This study aimed to compare the analgesic effects of programmed intermittent epidural boluses (PIEB) and continuous epidural infusion (CEI) for postoperative analgesia after elective cesarean section (CS). Methods Seventy-four women who underwent elective CS were randomized to receive either PIEB or CEI. The PIEB group received 4 ml-intermittent boluses of 0.11% ropivacaine every hour at a rate of 120 ml/h. The CEI group received a constant rate of 4 ml/h of 0.11% ropivacaine. The primary outcome was the pain score at rest at 36 h after CS. Secondary outcomes included the pain scores during mobilization, time-weighted pain scores, the incidence of motor blockade, and complications-related epidural analgesia during 36 h after CS. Results The pain score at rest at 36 h after CS was significantly lower in the PIEB group compared with that in the CEI group (3.0 vs. 0.0; median difference: 2, 95% CI [1, 2], P < 0.001). The mean time-weighted pain scores at rest and during mobilizations were also significantly lower in the PIEB group than in the CEI group (pain at rest; mean difference [MD]: 37.5, 95% CI [24.6, 50.4], P < 0.001/pain during mobilization; MD: 56.6, 95% CI [39.8, 73.5], P < 0.001). The incidence of motor blockade was significantly reduced in the PIEB group compared with that in the CEI group (P < 0.001). Conclusions PIEB provides superior analgesia with less motor blockade than CEI in postpartum women after CS, without any apparent adverse events.

Background: In healthy dentin conditions, odontoblasts have an important role such as protection from invasion of pathogens. In mammalian teeth, progenitors such as mesenchymal stem cells (MSCs) can migrate and differentiate into odontoblast-like cells, leading to the formation of reparative dentin. For differentiation using stem cells, it is crucial to provide conditions similar to the complex and intricate in vivo environment. The purpose of this study was to evaluate the potential of differentiation into odonto/osteoblasts, and compare co-culture with/without epithelial cells. Methods: MSCs and epithelial cells were successfully isolated from dental tissues. We investigated the influences of epithelial cells on the differentiation process of dental pulp stem cells into odonto/osteoblasts using co-culture systems. The differentiation potential with/without epithelial cells was analyzed for the expression of specific markers and calcium contents. Results: Differentiated odonto/osteoblast derived from dental pulp tissue-derived mesenchymal stem cells with/without epithelial cells were evaluated by qRT-PCR, immunostaining, calcium content, and ALP staining. The expression of odonto/osteoblast-specific markers, calcium content, and ALP staining intensity were significantly increased in differentiated cells. Moreover, the odonto/osteogenic differentiation capacity with epithelial cells co-culture was significantly higher than without epithelial cells co-culture. Conclusions: These results suggest that odonto/osteogenic differentiation co-cultured with epithelial cells has a more efficient application.

Fibroblasts are cells that reside within the fibrous or loose connective tissues of most mammalian organs. For research purposes, fibroblasts are often subjected to long-term culture under defined conditions, during which their properties can significantly change. It is essential to understand and document these changes to obtain reliable outcomes. For the quantification of specific gene expressions, the most reliable and widely used technique is quantitative real-time polymerase chain reaction (qRT-PCR). Here, we assessed the impact of a reference gene’s stability on a qRT-PCR analysis of long-term cultured canine skin fibroblasts. After successfully isolating the fibroblasts from canine skin tissues, they were cultured and evaluated for proliferation and β-galactosidase activity at different passage numbers. With extended culture, the fibroblasts showed a long doubling time and elevated β-galactosidase activity. Using three widely used algorithms, geNorm, Normfinder, and Bestkeeper, we identified HPRT1, YWHAZ, and GUSB as the most stable reference genes for both early- and late-passage fibroblasts. Conventional reference genes such as GAPDH were found to be less stable than those genes. The normalization of Vimentin by the stable genes showed statistical differences, whereas normalization by an unstable gene did not. Collectively, this study indicates that using stable reference genes is essential for accurately and reliably measuring gene expression in both early- and late-passage fibroblasts. These findings provide valuable insights into internal controls for gene expression studies and are expected to be utilized for analyzing gene expression patterns in molecular biology research.

Propagation of transgenic animals by germline transmission using assisted reproductive technologies such as in vitro fertilization (IVF) is the most efficient way to produce transgenic colonies for biomedical research. The objective of this study was to generate transgenic puppies from a founder dog expressing the mutated human amyloid precursor protein (mhAPP) gene. Experiment I assessed the characteristics of the semen prepared by freshly diluted, swim-up, and Percoll gradient methods using a computer-assisted semen analyzer (CASA). Motile and progressively motile sperm counts were higher in the Percoll gradient samples (p

Objective The present study analyzed the influence of co-transferring embryos with high and low cloning efficiencies produced via somatic cell nuclear transfer (SCNT) on pregnancy outcomes in dogs. Methods Cloned dogs were produced by SCNT using donor cells derived from a Tibetan Mastiff (TM) and Toy Poodle (TP). The in vivo developmental capacity of cloned embryos was evaluated. The pregnancy and parturition rates were determined following single transfer of 284 fused oocytes into 21 surrogates and co-transfer of 47 fused oocytes into four surrogates. Results When cloned embryos produced using a single type of donor cell were transferred into surrogates, the pregnancy and live birth rates were significantly higher following transfer of embryos produced using TP donor cells than following transfer of embryos produced using TM donor cells. Next, pregnancy and live birth rates were compared following single and co-transfer of these cloned embryos. The pregnancy and live birth rates were similar upon co-transfer of embryos and single transfer of embryos produced using TP donor cells but were significantly lower upon single transfer of embryos produced using TM donor cells. Furthermore, the parturition rate for TM dogs and the percentage of these dogs that remained alive until weaning was significantly higher upon co-transfer than upon single transfer of embryos. However, there was no difference between the two embryo transfer methods for TP dogs. The mean birth weight of cloned TM dogs was significantly higher upon single transfer than upon co-transfer of embryos. However, the body weight of TM dogs did not significantly differ between the two embryo transfer methods after day 5. Conclusion For cloned embryos with a lower developmental competence, the parturition rate and percentage of dogs that remain alive until weaning are increased when they are co-transferred with cloned embryos with a greater developmental competence.

Abstract Animal cloning has been popularized for more than two decades, since the birth of Dolly the Sheep 25 years ago in 1996. There has been an apparent waning of interest in cloning, evident by a reduced number of reports. Over 1500 dogs, representing approximately 20% of the American Kennel Club’s recognized breeds, have now been cloned, making the dog (Canis familiaris) one of the most successfully cloned mammals. Dogs have a unique relationship with humans, dating to prehistory, and a high degree of genome homology to humans. A number of phenotypic variations, rarely recorded in natural reproduction have been observed in in these more than 1000 clones. These observations differ between donors and their clones, and between clones from the same donor, indicating a non-genetic effect. These differences cannot be fully explained by current understandings but point to epigenetic and cellular reprograming effects of somatic cell nuclear transfer. Notably, some phenotypic variations have been reversed through further cloning. Here we summarize these observations and elaborate on the cloning procedure.

Objective The present study evaluated the efficiency of embryo development and pregnancy of somatic cell nuclear transfer (SCNT) embryos using different source-matured oocytes in Camelus dromedarius. Methods Camelus dromedarius embryos were produced by SCNT using in vivo- and in vitro- matured oocytes. In vitro embryo developmental capacity of reconstructed embryos was evaluated. To confirm the efficiency of pregnancy and live birth rates, a total of 72 blastocysts using in vitro- matured oocytes transferred into 45 surrogates and 95 blastocysts using in vivo- matured oocytes were transferred into 62 surrogates by transvaginal method. Results The collected oocytes derived from ovum pick up showed higher maturation potential into metaphase II oocytes than oocytes from the slaughterhouse. The competence of cleavage, and blastocyst were also significantly higher in in vivo- matured oocytes than in vitro- matured oocytes. After embryo transfer, 11 pregnant and 10 live births were confirmed in in vivo- matured oocytes group, and 2 pregnant and 1 live birth were confirmed in in vitro- matured oocytes group. Furthermore, blastocysts produced by in vivo-matured oocytes resulted in significantly higher early pregnancy and live birth rates than in vitro-matured oocytes. Conclusion In this study, SCNT embryos using in vivo- and in vitro-matured camel oocytes were successfully developed, and pregnancy was established in recipient camels. We also confirmed that in vivo-matured oocytes improved the development of embryos and the pregnancy capacity using the blastocyst embryo transfer method.

A new flavan-3-ol glycoside, compound 1 (careyarboside A), was isolated from the ethanol extract of the stems of Careya arborea, together with six known compounds. Of these, three phenolic glycosides (leonuriside A, picraquassioside D, and careyarboside B) have never been previously isolated from genus Careya and family Lecythidaceae. Their chemical structures were identified using spectroscopic data, specifically HRESIMS, 1D and 2D NMR data, including 1H-1H COSY, HSQC, and HMBC. In addition, the chemical structures of the known compounds were identified through spectral comparisons with those of published values.

Background: Individuals with forward head posture (FHP) have neck pain. To correct the FHP, a posture correction band is commonly used. However, we do not know the posture correction band influenced the pulmonary function in individuals with FHP. Objects: This study aimed to elucidate the effects of the posture correction band on the pulmonary function in young adults with neck pain and FHP and to monitor how the pulmonary function changed over time. Methods: Twenty subjects with chronic neck pain and forward head posture were recruited. Subjects performed pulmonary function test four times: before, immediately, and 2 hours after wearing the postural band, and immediately after undressing the postural band. Vital capacity (VC), forced vital capacity (FVC), peak expiratory flow (PEF), and forced expiratory volume at one second (FEV1) were measured. The modified Borg dyspnea scale was used to measure each subject’s responses to the posture correction band. The mixed-effect linear regression was used to the effect of the posture correction band over time. Results: There were no significant differences in VC, FVC, PEF, FEV1 values over time (p > 0.05), although all values slightly decreased after applying posture correction band. However, the score of the modified Borg scale significantly changed after wearing the postural bands (p < 0.05), indicating the subject felt discomfort with posture correction band during breathing. Conclusion: Because the posture correction band did not change the pulmonary function over time, but it induces psychological discomforts during breathing in people with FHP. Therefore, this posture correction band can be used for FHP realignment after discussion with the subjects.

Abstract Background Transverse facial clefts are Tessier’s number 7 facial cleft among numbers 1–15 in Tessier’s classification of craniofacial malformations, which varies from a simple widening oral commissure to a complete fissure extending towards the external ear. Case presentation In a patient with a transverse facial cleft, to functionally arrange the orbicularis oris muscle and form the oral commissure naturally, we performed a surgical procedure including orbicularis oris muscle reconstruction and cheiloplasty with Z-plasty. Conclusion We achieved good results functionally and esthetically by orbicularis oris muscle reconstruction and cheiloplasty with Z-plasty. The surgical modality of our anatomical repair and 3 months follow-up results are presented.

Abstract Background Genetic engineering technology such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides a powerful tool for developing disease models and determining gene functions. Recent interests in canine cancer models have highlighted the necessity of developing genetic engineering tools for dogs. In this study, we attempted to generate optimized CRISPR/Cas9 system to target canine tumor protein 53 (TP53), one of the most crucial tumor suppressor genes, to establish TP53 knockout canine cells for canine cancer research. Results We constructed CRISPR/Cas9 vectors using each of three TP53 gene-targeting guide RNAs (gRNAs) with minimal off-target potential. After transfection, we obtained several clones of TP53 knockout cells containing “indel” mutations in the targeted locus which had infinite cellular life span, resistance to genotoxicity, and unstable genomic status in contrast to normal cells. Of the established TP53 knockout cells, TP53KO#30 cells targeted by TP53 gRNA #30 showed non-cancerous phenotypes without oncogenic activation both in vitro and in vivo. More importantly, no off-target alteration was detected in TP53KO#30 cells. We also tested the developmental capacity of TP53 knockout cells after application of the somatic cell nuclear transfer technique. Conclusions Our results indicated that TP53 in canine cells was effectively and specifically targeted by our CRISPR/Cas9 system. Thus, we suggest our CRISPR/Cas9-derived canine TP53 knockout cells as a useful platform to reveal novel oncogenic functions and effects of developing anti-cancer therapeutics.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to reproduce animals with superior genetics. Viable cell lines are usually established from tissues collected by biopsy from living animals in the SCNT program. In the present study, tissues were collected and preserved from a suddenly deceased champion camel. We established cell lines from these decade-old tissues and used them as nuclear donors. After 42 h of in vitro maturation, 68.00 ± 2.40% of oocytes reached the metaphase II (M II) stage while 87.31 ± 2.57% in vivo collected oocytes were matured at collection (p < 0.05). We observed a higher blastocyst formation rate when in vivo matured oocytes (43.45 ± 2.07%) were used compared to in vitro matured oocytes (21.52 ± 1.74%). The live birth rate was 6.45% vs. 16.67% for in vitro and in vivo matured oocytes, respectively. Microsatellite analysis of 13 camel loci revealed that all the SCNT-derived offspring were identical to each other and with their somatic cell donor. The present study succeeded in the resurrection of 11 healthy offspring from the decade-old vitrified tissues of a single somatic cell donor individual using both in vitro and in vivo matured oocytes.

Granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine, belongs to the hematopoietic growth factor family. Recent studies have reported that G-CSF is a predictive biomarker of oocyte and embryo developmental competence in humans. The aim of our study was to determine whether CSF3 and its receptor (CSF3R) were expressed in porcine maternal reproductive tissues (oviduct and uterus), cumulus cells, and embryos and to investigate the effects of human recombinant G-CSF (hrG-CSF) supplementation during in vitro culture (IVC) on the developmental competence of pre-implantation embryos. To do this, we first performed reverse-transcription polymerase chain reaction (RT-PCR). Second, we performed parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) to evaluate the embryonic developmental potential after hrG-CSF supplementation based on various concentrations (0 ng/mL, 10 ng/mL, 50 ng/mL, and 100 ng/mL) and durations (Un-treated, Days 0-3, Days 4-7, and Days 0-7) of IVC. Finally, we examined transcriptional levels of several marker genes in blastocysts. The results of our study showed that CSF3 transcript was present in all samples we assessed. CSF3-R was also detected, except in cumulus cells and blastocysts from PA. Furthermore, 10 ng/mL and Days 0-7 were the optimal concentration and duration for the viability of in vitro embryonic development, especially for SCNT-derived embryos. The rate of blastocyst formation and the total cell number of blastocysts were significantly enhanced, while the number and index of apoptotic nuclei were significantly decreased in optimal condition groups compared to others. Moreover, the transcriptional levels of anti-apoptotis- (BCL2), proliferation- (PCNA), and pluripotency- (POU5F1) related genes were dramatically upregulated. In conclusion, for the first time, we demonstrated that CSF3 and CSF3R were expressed in porcine reproductive organs, cells, and embryos. Additionally, we determined that hrG-CSF treatment improved porcine embryonic development capacity in vitro.

Recent advances in somatic cell nuclear transfer (SCNT) in canines facilitate the production of canine transgenic models. Owing to the importance of stable and strong promoter activity in transgenic animals, we tested human elongation factor 1α (hEF1α) and cytomegalovirus (CMV) promoter sequences in SCNT transgenic dogs. After transfection, transgenic donor fibroblasts with the hEF1α-enhanced green fluorescence protein (EGFP) transgene were successfully isolated using fluorescence-activated cell sorting (FACS). We obtained four puppies, after SCNT, and identified three puppies as being transgenic using PCR analysis. Unexpectedly, EGFP regulated by hEF1α promoter was not observed at the organismal and cellular levels in these transgenic dogs. EGFP expression was rescued by the inhibition of DNA methyltransferases, implying that the hEF1α promoter is silenced by DNA methylation. Next, donor cells with CMV-EGFP transgene were successfully established and SCNT was performed. Three puppies of six born puppies were confirmed to be transgenic. Unlike hEF1α-regulated EGFP, CMV-regulated EGFP was strongly detectable at both the organismal and cellular levels in all transgenic dogs, even after 19 months. In conclusion, our study suggests that the CMV promoter is more suitable, than the hEF1α promoter, for stable transgene expression in SCNT-derived transgenic canine model.

Mesenchymal stem cells (MSCs) are promising multipotent cells with applications for cartilage tissue regeneration in stem cell-based therapies. In cartilage regeneration, both bone marrow (BM-MSCs) and synovial fluid (SF-MSCs) are valuable sources. However, the cellular characteristics and chondrocyte differentiation potential were not reported in either of the camel stem cells. The in vitro chondrocyte differentiation competence of MSCs, from (BM and SF) sources of the same Camelus dromedaries (camel) donor, was determined. Both MSCs were evaluated on pluripotent markers and proliferation capacity. After passage three, both MSCs showed fibroblast-like morphology. The proliferation capacity was significantly increased in SF-MSCs compared to BM-MSCs. Furthermore, SF-MSCs showed an enhanced expression of transcription factors than BM-MSCs. SF-MSCs exhibited lower differentiation potential toward adipocytes than BM-MSCs. However, the osteoblast differentiation potential was similar in MSCs from both sources. Chondrogenic pellets obtained from SF-MSCs revealed higher levels of chondrocyte-specific markers than those from BM-MSCs. Additionally, glycosaminoglycan (GAG) content was elevated in SF-MSCs related to BM-MSCs. This is, to our knowledge, the first study to establish BM-MSCs and SF-MSCs from the same donor and to demonstrate in vitro differentiation potential into chondrocytes in camels.

Mesenchymal stem cells (MSCs) showed in vitro mesoderm-lineage differentiation and self-renewal capacity. However, no comparative study was reported on the biological characteristics of stem cells derived from skeletal muscle (SM-MSCs), dermal skin (DS-MSCs), and adipose tissues (A-MSCs) from a single donor in camels. The present study aimed to evaluate the influence of MSCs source on stem cell characteristics. We evaluated proliferation capacity and mesoderm-lineage differentiation potential from SM-MSCs, DS-MSCs, and A-MSCs. They showed spindle-like morphology after homogenization. The proliferation ability was not significantly difference in any of the groups. Furthermore, the portion of the cell cycle and expression of pluripotent markers (Oct4, Sox2, and Nanog) were similar in all cell lines at passage 3. The differentiation capacity of A-MSCs into adipocytes was significantly higher than that of SM-MSCs and DS-MSCs. However, the osteoblast differentiation capacity of A-MSCs was significantly lower than that of SM-MSCs and DS-MSCs. Additionally, after osteoblast differentiation, the alkaline phosphatase (ALP) activity and calcium content significantly decreased in A-MSCs compared to SM-MSCs and DS-MSCs. To the best of our knowledge, we primarily established MSCs from the single camel and demonstrated their comparative characteristics, including expression of pluripotent factors and proliferation, and in vitro differentiation capacity into adipocytes and osteoblasts.

The fast pyrolysis of coffee ground for bio-crude oil production was simulated in a tilted-slide reactor. The biochemical composition was derived by an extended biomass characterization method based on the elemental analysis. The simulation was performed in a steady-state and a Lagrangian multiphase model was adopted to describe the transport of sand and biomass particles together with a multistep kinetic mechanism for fast pyrolysis. When the secondary tar cracking reactions were not considered the volatile yield increased monotonically with temperature. The inclusion of secondary reactions could improve the prediction of volatile yield which turn to decrease at higher temperature. It was found that not only the maximum volatile yield but also the corresponding reactor temperature agreed well with the experimental results. At the temperature higher than 550 °C the trend of volatile yield is similar to that of experiment while it is larger at lower reactor temperature. The individual species yields were compared at various reactor temperatures and the pyrolysis processes were analyzed by tracking the reference components when they were decomposed along the distance. It was found that the reactor temperature should be above 500 °C for effective pyrolysis of all reference components of coffee ground.

Canine induced pluripotent stem cells (ciPSCs) can provide great potential for regenerative veterinary medicine. Several reports have described the generation of canine somatic cell-derived iPSCs; however, none have described the canine somatic cell reprogramming using a non-integrating and self-replicating RNA transfection method. The purpose of this study was to investigate the optimal strategy using this approach and characterize the transition stage of ciPSCs. In this study, fibroblasts obtained from a 13-year-old dog were reprogrammed using a non-integrating Venezuelan equine encephalitis (VEE) RNA virus replicon, which has four reprogramming factors (collectively referred to as T7-VEE-OKS-iG and comprised of hOct4, hKlf4, hSox2, and hGlis1) and co-transfected with the T7-VEE-OKS-iG RNA and B18R mRNA for 4 h. One day after the final transfection, the cells were selected with puromycin (0.5 µg/mL) until day 10. After about 25 days, putative ciPSC colonies were identified showing TRA-1-60 expression and alkaline phosphatase activity. To determine the optimal culture conditions, the basic fibroblast growth factor in the culture medium was replaced with a modified medium supplemented with murine leukemia inhibitory factor (mLIF) and two kinase inhibitors (2i), PD0325901(MEK1/2 inhibitor) and CHIR99021 (GSK3β inhibitor). The derived colonies showed resemblance to naïve iPSCs in their morphology (dome-shaped) and are dependent on mLIF and 2i condition to maintain an undifferentiated phenotype. The expression of endogenous pluripotency markers such as Oct4, Nanog, and Rex1 transcripts were confirmed, suggesting that induced ciPSCs were in the late intermediate stage of reprogramming. In conclusion, the non-integrating and self-replicating VEE RNA replicon system can potentially make a great contribution to the generation of clinically applicable ciPSCs, and the findings of this study suggest a new method to utilize the VEE RNA approach for canine somatic cell reprogramming.

Coffee ground has been recently considered as a new biomass resource in relation to the increasing coffee consumption worldwide. The bio-crude oil can be produced by fast pyrolysis of coffee ground, and it has advantages of larger heating values in comparison with those from other biomass. But the bio-crude oil from coffee ground has a significantly high viscosity which can hinder the application to conventional burners. In this study, a pilot-scale burner system with a 35 kW capacity with an air-blast atomizing nozzle was developed for the combustion of bio-crude oil from coffee ground with a high viscosity. A downward fuel injection system was adopted to enhance the ignition of fuel spray and the flame stabilization, and a movable block swirl generator was installed for the combustion air. The bio-crude oil was blended with ethanol at the volumetric ratio of 9:1 to enhance the combustion characteristics. The effect of various atomizing air pressures, swirl intensities, and overall equivalence ratios on the flame stability and gaseous emission were investigated to find out the optimum operating conditions for a bio-crude oil burner.

Veratrum spp. have traditionally been used in folk medicine to treat various pathologies. In this study, nine compounds, comprising one simple phenolic compound (1), three stilbenoids (2-4), and five flavonoids (5-9), were isolated from the aerial parts of Veratrum versicolor f. viride Nakai. The structures of these compounds were elucidated by spectroscopic analyses and comparison with reported data. Together, all reported compounds were isolated from V. versicolor f. viride for the first time in the study. Among them, two flavone aglycone tricetins (7 and 9) have never been isolated from the genus Veratrum or the family Melanthiaceae. The ethanol extract and isolated compounds were assessed for their inhibitory effects on elastase, tyrosinase, and melanin synthesis. Compounds 5 and 7 inhibited elastase (IC50: 292.25 ± 14.39 and 800.41 ± 5.86 µM, respectively), whereas compounds 2-5 inhibited tyrosinase with IC50 values in the range of 6.42 ~ 51.19 µM, respectively. In addition, compounds 3-6 and 8 exhibited dose-dependent inhibition (70.4% ~ 91.0%) of melanogenesis at a concentration of 100 µM. [ABSTRACT FROM AUTHOR]

Many different approaches have been developed to improve the efficiency of animal cloning by somatic cell nuclear transfer (SCNT), one of which is to modify histone acetylation levels using histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA). In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from SCNT. We found that TSA treatment (50 nM) for 24 h following oocyte activation improved blastocyst formation rates (to 22.0%) compared with 8.9% in the non-treatment group and total cell number of the blastocysts for determining embryo quality also increased significantly (88.9→114.4). Changes in histone acetylation levels as a result of TSA treatment were examined using indirect immunofluorescence and confocal microscopy scanning. Results showed that the histone acetylation level in TSA-treated embryos was higher than that in controls at both acetylated histone H3 lysine 9 (AcH3K9) and acetylated histone H4 lysine 12 (AcH4K12). Next, we compared the expression patterns of seven genes (OCT4, ID1; the pluripotent genes, H19, NNAT, PEG1; the imprinting genes, cytokeratin 8 and 18; the trophoblast marker genes). The SCNT blastocysts both with and without TSA treatment showed lower levels of OCT4, ID1, cytokeratin 8 and 18 than those of the in vivo blastocysts. In the case of the imprinting genes H19 and NNAT, except PEG1, the SCNT blastocysts both with and without TSA treatment showed higher levels than those of the in vivo blastocysts. Although the gene expression patterns between cloned blastocysts and their in vivo counterparts were different regardless of TSA treatment, it appears that several genes in NT blastocysts after TSA treatment showed a slight tendency toward expression patterns of in vivo blastocysts. Our results suggest that TSA treatment may improve preimplantation porcine embryo development following SCNT.

The present study was conducted to generate transgenic pigs coexpressing human CD55, CD59, and H-transferase (HT) using an IRES-mediated polycistronic vector. The study focused on hyperacute rejection (HAR) when considering clinical xenotransplantation as an alternative source for human organ transplants. In total, 35 transgenic cloned piglets were produced by somatic cell nuclear transfer (SCNT) and were confirmed for genomic integration of the transgenes from umbilical cord samples by PCR analysis. Eighteen swine umbilical vein endothelial cells (SUVEC) were isolated from umbilical cord veins freshly obtained from the piglets. We observed a higher expression of transgenes in the transgenic SUVEC (Tg SUVEC) compared with the human umbilical vein endothelial cells (HUVEC). Among these genes, HT and hCD59 were expressed at a higher level in the tested Tg organs compared with non-Tg control organs, but there was no difference in hCD55 expression between them. The transgenes in various organs of the Tg clones revealed organ-specific and spatial expression patterns. Using from 0 to 50% human serum solutions, we performed human complement-mediated cytolysis assays. The results showed that, overall, the Tg SUVEC tested had greater survival rates than did the non-Tg SUVEC, and the Tg SUVEC with higher HT expression levels tended to have more down-regulated α-Gal epitope expression, resulting in greater protection against cytotoxicity. By contrast, several Tg SUVEC with low CD55 expression exhibited a decreased resistance response to cytolysis. These results indicated that the levels of HT expression were inversely correlated with the levels of α-Gal epitope expression and that the combined expression of hCD55, hCD59, and HT proteins in SUVECs markedly enhances a protective response to human serum-mediated cytolysis. Taken together, these results suggest that combining a polycistronic vector system with SCNT methods provides a fast and efficient alternative for the generation of transgenic large animals with multiple genetic modifications.