CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts.

The multi-gene editing porcine cell model can analyze the genetic mechanisms of multiple genes, which is beneficial for accelerating genetic breeding. However, there has been a lack of an effective strategy to simultaneously perform precise multi-gene editing in porcine cells. In this study, we aimed to improve the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells. CRISPR RNP, including Cas9 protein, sgRNA, and ssODN, was used to generate precise nucleotide substitutions by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by PCR for each target site. To enhance HDR efficacy, small-molecule M3814 and phosphorothioate-modified ssODN were employed. All target DNA samples were sequenced and analyzed, and the efficiencies of different combinations of the CRISPR RNP system in target sites were compared. The results showed that when 2 µM M3814, a small molecule which inhibits NHEJ-mediated repair by blocking DNA-PKs activity, was used, there was no toxicity to PFFs. The CRISPR RNP-mediated HDR efficiency increased 3.62-fold. The combination of CRISPR RNP with 2 µM M3814 and PS-ssODNs achieved an HDR-mediated precision gene modification efficiency of approximately 42.81% in mutated cells, a 6.38-fold increase compared to the control group. Then, we used the optimized CRISPR RNP system to perform simultaneous editing of two and three loci at the INS and RLN3 genes. The results showed that the CRISPR RNP system could simultaneously edit two and three loci. The efficiency of simultaneous editing of two loci was not significantly different from that of single-gene editing compared to the efficiency of single-locus editing. The efficiency of simultaneous precise editing of INS, RLN3 exon 1, and RLN3 exon 2 was 0.29%, 0.24%, and 1.05%, respectively. This study demonstrated that a 2 µM M3814 combination with PS-ssODNs improves the efficacy of CRISPR RNP-mediated precise gene editing and allows for precise editing of up to three genes simultaneously in porcine cells.

SINE-Associated LncRNA SAWPA Regulates Porcine Zygotic Genome Activation.

In mice, retrotransposon-associated long noncoding RNAs (lncRNA) play important regulatory roles in pre-implantation development; however, it is largely unknown whether they function in the pre-implantation development in pigs. The current study aims to screen for retrotransposon-associated lncRNA in porcine early embryos and identifies a porcine 8-cell embryo-specific SINE-associated nuclear long noncoding RNA named SAWPA. SAWPA is essential for porcine embryonic development as depletion of SAWPA results in a developmental arrest at the 8-cell stage, accompanied by the inhibition of the JNK-MAPK signaling pathway. Mechanistically, SAWPA works in trans as a transcription factor for JNK through the formation of an RNA-protein complex with HNRNPA1 and MED8 binding the SINE elements upstream of JNK. Therefore, as the first functional SINE-associated long noncoding RNAs in pigs, SAWPA provides novel insights for the mechanism research on retrotransposons in mammalian pre-implantation development.

Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript.

Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem-loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications.

Multiplexed genome engineering for porcine fetal fibroblasts with gRNA-tRNA arrays based on CRISPR/Cas9.

Multiplex gene modifications are highly required for various fields of porcine research. In many species, the CRISPR/Cas9 system has been widely applied for genomic editing and provides a potential tool for introducing multiplex genome mutations simultaneously. Here, we present a CRISPR-Cas9 gRNA-tRNA array (GTR-CRISPR) for multiplexed engineering of porcine fetal fibroblasts (PFFs). We successfully produced multiple sgRNAs using only one Pol III promoter by taking advantage of the endogenous tRNA processing mechanism in porcine cells. Using an all-in-one construct carrying GTR and Cas9, we disrupted the IGFBP3, MSTN, MC4R, and SOCS2 genes in multiple codon regions in one PFF cell simultaneously. This technique allows the simultaneous disruption of four genes with 5.5% efficiency. As a result, this approach may effectively target multiple genes at the same time, making it a powerful tool for establishing multiple genes mutant cells in pigs.

Tannin Reduces the Incidence of Polyspermic Penetration in Porcine Oocytes.

Tannin (TA) improves porcine oocyte cytoplasmic maturation and subsequent embryonic development after in vitro fertilization (IVF). However, the mechanism through which TA blocks polyspermy after IVF remains unclear. Hence, the biological function of organelles (cortical granule [CG], Golgi apparatus, endoplasmic reticulum [ER], and mitochondria) and the incidence of polyspermic penetration were examined. We found no significant difference in oocyte nuclear maturation among the 1 µg/mL, 10 µg/mL TA, and control groups. Moreover, 100 µg/mL TA significantly reduced 1st polar body formation rate compared to the other groups. Additionally, 1 and 10 µg/mL TA significantly increased the protein levels of GDF9, BMP15, and CDK1 compared to the control and 100 µg/mL TA groups. Interestingly, 1 and 10 µg/mL TA improved the normal distribution of CGs, Golgi, ER, and mitochondria by upregulating organelle-related gene expression and downregulating ER stress (CHOP) gene expression. Simultaneously, 1 and 10 µg/mL TA significantly increased the proportion of normal fertilized oocytes (2 pronuclei; 2 PN) and blastocyst formation rate compared to the control, as well as that of 100 µg/mL TA after IVF by upregulating polyspermy-related genes. In conclusion, TA during IVM enhances 2PN and blastocyst formation rates by regulating organelles' functions and activities.

Ramelteon Reduces Oxidative Stress by Maintenance of Lipid Homeostasis in Porcine Oocytes.

This study aimed to determine the underlying mechanism of ramelteon on the competence of oocyte and subsequent embryo development in pigs during in vitro maturation (IVM). Our results showed that the cumulus expansion index was significantly lower in the control group compared to the ramelteon groups (p < 0.05). Moreover, supplementation of 10−11 and 10−9 M ramelteon significantly increased the cumulus expansion and development-related genes expression, and reduced apoptosis in cumulus cells (p < 0.05). In oocytes, the nuclear maturation rate was significantly improved in 10−11, 10−9, and 10−7 M ramelteon groups compared to the control (p < 0.05). Additionally, the level of intracellular GSH was significantly increased and ROS was significantly decreased in ramelteon-supplemented groups, and the gene expression of oocyte development and apoptosis were significantly up- and down-regulated by 10−11 and 10−9 M ramelteon (p < 0.05), respectively. The immunofluorescence results showed that the protein levels of GDF9, BMP15, SOD1, CDK1, and PGC1α were significantly increased by 10−11 M ramelteon compared to the control (p < 0.05). Although there was no significant difference in cleavage rate, the blastocyst formation rate, total cell numbers, and hatching/-ed rate were significantly improved in 10−11 M ramelteon group compared to the control (p < 0.05). Furthermore, embryo development, hatching, and mitochondrial biogenesis-related genes were dramatically up-regulated by 10−11 M ramelteon (p < 0.05). In addition, the activities of lipogenesis and lipolysis in oocytes were dramatically increased by 10−11 M ramelteon compared to the control (p < 0.05). In conclusion, supplementation of 10−11 M ramelteon during IVM improved the oocyte maturation and subsequent embryo development by reducing oxidative stress and maintenance of lipid homeostasis.

Melatonin Regulates Lipid Metabolism in Porcine Cumulus-Oocyte Complexes via the Melatonin Receptor 2.

Previous studies suggest that the inclusion of melatonin (MTn) in in vitro maturation protocols improves the developmental competence of oocytes by scavenging reactive oxygen species (ROS). However, the molecular mechanisms integrating melatonin receptor (MT)-mediated lipid metabolism and redox signaling during in vitro cumulus-oocyte complex (COC) development still remain unclear. Here, we aimed to elucidate the potential role of MTn receptors in lipid metabolic adjustments during in vitro porcine COC development. We observed that MTn-mediated Gsα-cAMP/PKA signaling facilitated lipolysis primarily through the MT2 receptor and subsequently increased fatty acid (FA) release by hydrolyzing intracellular triglycerides (TGs) in cumulus cells. Furthermore, CD36 was a critical FA transporter that transported available FAs from cumulus cells to oocytes and promoted de novo TG synthesis in the latter. In addition, MTn regulated lipogenesis and intracellular lipolysis to maintain lipid homeostasis and limit ROS production, thereby supporting oocyte cytoplasmic maturation and the subsequent embryo development. Taken together, these findings provide insight into the possible mechanism integrating MT2-mediated lipid homeostasis and redox signaling, which limits ROS production during in vitro COC development. Therefore, understanding the dynamics of the interactions between lipid homeostasis and redox signaling driven by MT2 is necessary in order to predict drug targets and the effects of therapeutics used to improve female reproductive health.

Tannin Supplementation Improves Oocyte Cytoplasmic Maturation and Subsequent Embryo Development in Pigs.

To investigate the effects of tannins (TA) on porcine oocyte in vitro maturation (IVM), different concentrations of TA (0, 1, 10 and 100 µg/mL) were supplemented with a maturation medium and the COCs and subsequent embryonic development were examined. The results showed that 10 µg/mL TA significantly improved the cumulus expansion index (CEI), cumulus-expansion-related genes (PTGS1, PTGS2, PTX-3, TNFAIP6 and HAS2) expression and blastocyst formation rates after parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) compared to the control groups, but not oocyte nuclear maturation. Nevertheless, 10 µg/mL TA dramatically enhanced the mRNA expression of oocyte-development-related genes (BMP15, GDF9, CDC2 and CYCLIN B1), GSH, ATP, SOD1, PGC1α, BMP15, GDF9 and CDC2 levels and reduced intracellular ROS level in porcine oocytes. These results indicated that porcine oocyte cytoplasmic maturation was improved by 10 µg/mL TA treatment during IVM. In contrast, a high concentration of TA (100 µg/mL) significantly decreased the CEI and PTGS1, PTGS2, PTX-3 and HAS2 mRNA expressions in cumulus cells, and reduced oocyte nuclear maturation and the total cell numbers/blastocyst. In general, these data showed that 10 µg/mL TA supplementation has beneficial effects on oocyte cytoplasmic maturation and subsequent embryonic development in pigs.

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.

Derivation of endothelial cells from porcine induced pluripotent stem cells by optimized single layer culture system.

Regenerative therapy holds great promise in the development of cures of some untreatable diseases such as cardiovascular diseases, and pluripotent stem cells (PSCs) including induced PSCs (iPSCs) are the most important regenerative seed cells. Recently, differentiation of human PSCs into functional tissues and cells in vitro has been widely reported. However, although porcine reports are rare they are quite essential, as the pig is an important animal model for the in vitro generation of human organs. In this study, we reprogramed porcine embryonic fibroblasts into porcine iPSCs (piPSCs), and differentiated them into cluster of differentiation 31 (CD31)-positive endothelial cells (ECs) (piPSC-derived ECs, piPS-ECs) using an optimized single-layer culture method. During differentiation, we observed that a combination of GSK3ß inhibitor (CHIR99021) and bone morphogenetic protein 4 (BMP4) promoted mesodermal differentiation, resulting in higher proportions of CD31-positive cells than those from separate CHIR99021 or BMP4 treatment. Importantly, the piPS-ECs showed comparable morphological and functional properties to immortalized porcine aortic ECs, which are capable of taking up low-density lipoprotein and forming network structures on Matrigel. Our study, which is the first trial on a species other than human and mouse, has provided an optimized single-layer culture method for obtaining ECs from porcine PSCs. Our approach can be beneficial when evaluating autologous EC transplantation in pig models.

Lineage specification and pluripotency revealed by transcriptome analysis from oocyte to blastocyst in pig.

The inner cell mass (ICM) in blastocyst is the origin of all somatic and germ cells in mammals and pluripotent stem cells (PSCs) in vitro. As the conserved principles between pig and human, here we performed comprehensive single-cell RNA-seq for porcine early embryos from oocyte to early blastocyst (EB). We show the specification of the ICM and trophectoderm in morula and the molecular signature of the precursors. We demonstrate the existence of naïve pluripotency signature in morula and ICM of EB, and the specific pluripotent genes and the activity of signalling pathways highlight the characteristics of the naïve pluripotency. We observe the absence of dosage compensation with respect to X-chromosome (XC) in morula, and incomplete dosage compensation in the EB. However, the dynamics of dosage compensation may be independent of the expression of XIST induced XC inactivation. Our study describes molecular landmarks of embryogenesis in pig that will provide a better strategy for derivation of porcine PSCs and improve research in regenerative medicine.

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.

The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells.

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.

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.

A novel chemically defined serum- and feeder-free medium for undifferentiated growth of porcine pluripotent stem cells.

Development and improvement of in vitro culture system supporting self-renewal and unlimited proliferation of porcine pluripotent stem cells (pPSCs) is an indispensable process for the naïve pPSCs establishment. In this study, we modified the previous culture system and attempted to develop a novel chemically defined medium (KOFL) for the establishment of pPSCs. It has been cultured >45 passages with flat colony morphology and normal karyotypes in in vitro environment. These cells exhibited alkaline phosphatase activity and expressed pluripotency markers such as OCT4, SOX2, and NANOG, and also possessed differentiation abilities both in vitro and in vivo, proving by the formation of embryonic bodies and teratomas into three germ layers. Then the cells transfected with a green fluorescent protein (GFP) and the GFP positive cells contribute to the porcine preimplantation embryo development. In addition, these cells maintained long duration under feeder-free condition. In conclusion, our results demonstrated that the pPSCs could be derived from preimplantation porcine embryos in serum-free medium and cultured under the feeder-free condition, providing an effective reference for further optimization of the pPSCs culture system.

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.

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.

Synergistic effects of resveratrol and melatonin on in vitro maturation of porcine oocytes and subsequent embryo development.

Resveratrol and melatonin are known for their antioxidant properties and have various biological activities. The fact that they exhibit possible synergistic effects in phytomedicine researches suggests the use of a combination of these agents to promote porcine in vitro maturation (IVM) of oocytes. Therefore, we investigated the effects of resveratrol and/or melatonin on this process; cumulus-oocyte complexes underwent IVM culture with four different conditions (control, resveratrol, melatonin or their combination). Cumulus expansion, oocyte nuclear maturation and subsequent embryo development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) were evaluated. In experiment 1, all treatment groups significantly increased the proportion of complete cumulus expansion (degree 4) compared to the control, showing no difference among the treatment groups (P = 0.30). In experiment 2, oocytes matured with resveratrol and the combination had significantly higher metaphase-II (MII) rates than the control and melatonin groups, showing the highest (P < 0.05) MII rates in the combination group. In experiment 3, all treatment groups significantly increased blastocyst formation rates and total blastocyst cell numbers after PA compared to the control, but especially the combination showed the highest (P < 0.05) total cell numbers. In experiment 4, we selected the combination as the optimal condition and used this IVM system prior to SCNT. The combination treatment showed a significant (P < 0.05) increase in blastocyst formation rate and total cell numbers after SCNT. In conclusion, our results suggest that the combination of resveratrol and melatonin supported a synergistic increase in oocyte nuclear maturation and total cell numbers of PA blastocysts and improved the development of SCNT embryos.

Establishment and identification of cell lines from type O blood Korean native pigs and their efficiency in supporting embryonic development via somatic cell nuclear transfer.

Due to their similarities with humans in anatomy, physiology, and genetics miniature pigs are becoming an attractive model for biomedical research. We aim to establish and evaluate blood type O cells derived from Korean native pig (KNP), a typical miniature pig breed in Korea. Ten cell lines derived from 8 KNP piglets and one adult female KNP (kidney and ear tissues) were established. To confirm the presence of blood type O, genomic DNA, fucosyltransferase (FUT) expression, and immunofluorescence staining were examined. Additionally, fluorescence-activated cell sorting and somatic cell nuclear transfer were performed to investigate the normality of the cell lines and to evaluate their effectiveness in embryo development. We found no significant bands corresponding to specific blood group A, and no increase in FUT expression in cell lines derived from piglets No. 1, No. 4, No. 5, No. 8, and the adult female KNP; moreover, they showed normal levels of expression of α 1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase. There was no significant difference in embryo development between skin and kidney fibroblasts derived from the blood type O KNPs. In conclusion, we successfully established blood type O KNP cell lines, which may serve as a useful model in xenotransplantation research.