Dong-Il Jin

Sequence‐based characterization of the eight SLA loci in Korean native pigs

Eight swine leucocyte antigen (SLA) gene (SLA‐1, SLA‐2, SLA‐3, SLA‐6, DRA, DRB1, DQA, DQB1) alleles were identified using sequence‐based typing method in three Korean native pigs used for breeding at the National Institute of Animal Science in Korea. Six new alleles in class I genes and three new alleles in class II genes have been identified in this breed and can give valuable information for xenotransplantation and disease resistance.

Coordinated change of a ratio of methylated H3-Iysine 4 or acetylated H3 to acetylated H4 and DNA methylation is associated with tissue-specific gene expression in cloned pig

Various cell types in higher multicellular organisms are genetically homogenous, but are functionally and morphologically heterogeneous due to the differential expression of genes during development, which appears to be controlled by epigenetic mechanisms. However, the exact molecular mechanisms that govern the tissue-specific gene expression are poorly understood. Here, we show that dynamic changes in histone modifications and DNA methylation in the upstream coding region of a gene containing the transcription initiation site determine the tissue-specific gene expression pattern. The tissue-specific expression of the transgene correlated with DNA demethylation at specific CpG sites as well as significant changes in histone modifications from a low ratio of methylated H3- lysine 4 or acetylated H3-lysine 9, 14 to acetylated H4 to higher ratios. Based on the programmed status of transgene silenced in cloned mammalian ear-derived fibroblasts, the transgene could be reprogrammed by change of histone modification and DNA methylation by inhibiting both histone deacetylase and DNA methylation, resulting in high expression of the transgene. These findings indicate that dynamic change of histone modification and DNA methylation is potentially important in the establishment and maintenance of tissue-specific gene expression.

Treatment of fetal fibroblasts with DNA methylation inhibitors and/or histone deacetylase inhibitors improves the development of porcine nuclear transfer-derived embryos

This study investigated whether treating fetal fibroblast cells (donor cells) with epigenetic modification-inducing drugs could improve the development of porcine cloned embryos. Donor cells were treated with different DNA methylation inhibitors (5-aza-dC, zebularine or RG108; 5nM) or histone deacetylase inhibitors (TSA, NaBu or SCR; 50nM) for 1h, and then subjected to SCNT. All of the treated groups showed significantly higher blastocyst formation rates compared to the control group. We chose 5-aza-dC and TSA as a combined treatment, and found that donor cells co-treated with 2.5nM 5-aza-dC for 1h and subsequently treated with 50nM TSA for another 1h before SCNT showed significantly improved blastocyst rates compared to the control, 5-aza-dC-treated, and TSA-treated groups. The levels of DNA methylation were decreased (though not to a significant degree) in donor cells treated with 5-aza-dC, TSA or both. The histone H3 acetylation levels were significantly increased in donor cells treated with TSA or co-treated with 5-aza-dC and TSA. Donor cells simultaneously co-treated with 5nM 5-aza-dC and 50nM TSA for 1h showed increased apoptosis of SCNT blastocysts. However, when we decreased the concentration of 5-aza-dC to 2.5nM, the co-treatment induced less apoptosis among SCNT blastocysts and the blastocyst development rate improved. Together, these results indicate that treatment of donor cells with 5-aza-dC, TSA, or TSA plus a low dose of 5-aza-dC could improve the blastocyst development of porcine cloned embryos.

Comparison of PERV genomic locations between Asian and European pigs.

Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation.

Functional characterization of the ER stress induced X-box-binding protein-1 (Xbp-1) in the porcine system

BackgroundThe unfolded protein response (UPR) is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER) stress conditions. X-box-binding protein-1 (Xbp1) is a key transcription factor of UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The UPR induced by ER stress was extensively studied in diseases linked to protein misfolding and aggregations. However, in the porcine system, genes in the UPR pathway were not investigated. In this study, we isolated and characterized the porcine Xbp1 (pXbp1) gene in ER stress using porcine embryonic fibroblast (PEF) cells and porcine organs. ER stress was induced by the treatment of tunicamycin and cell viability was investigated by the MTT assay. For cloning and analyzing the expression pattern of pXbp1, RT-PCR analysis and Western blot were used. Knock-down of pXbp1 was performed by the siRNA-mediated gene silencing.ResultsWe found that the pXbp1 mRNA was the subject of the IRE1α-mediated unconventional splicing by ER stress. Knock-down of pXbp1 enhanced ER stress-mediated cell death in PEF cells. In adult organs, pXbp1 mRNA and protein were expressed and the spliced forms were detected.ConclusionsIt was first found that the UPR mechanisms and the function of pXbp1 in the porcine system. These results indicate that pXbp1 plays an important role during the ER stress response like other animal systems and open a new opportunity for examining the UPR pathway in the porcine model system.

Production of transgenic recloned piglets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer

We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.

Characterization of Porcine Endogenous Retrovirus Clones from the NIH Miniature Pig BAC Library

Pigs have been considered as donors for xenotransplantation in the replacement of human organs and tissues. However, porcine endogenous retroviruses (PERVs) might transmit new infectious disease to humans during xenotransplantation. To investigate PERV integration sites, 45 PERV-positive BAC clones, including 12 PERV-A, 16 PERV-B, and 17 PERV-C clones, were identified from the NIH miniature pig BAC library. The analysis of 12 selected full-length sequences of PERVs, including the long terminal repeat (LTR) region, identified the expected of open reading frame length, an indicative of active PERV, in all five PERV-C clones and one of the four PERV-B clones. Premature stop codons were observed in only three PERV-A clones. Also, eleven PERV integration sites were mapped using a 5000-rad IMpRH panel. The map locations of PERV-C clones have not been reported before, thus they are novel PERV clones identified in this study. The results could provide basic information for the elimination of site-specific PERVs in selection of pigs for xenotransplantation.

Abnormal Expression of TIMP‐2, SOD, Vimentin and PAI Proteins in Cloned Bovine Placentae

Cloned mammals suffer from high rates of placental abnormality and foetal loss during pregnancy. We previously used 2-D gel electrophoresis and mass spectrometry for global proteomic analysis of cloned and normal bovine placentae to identify differential protein expression patterns. Here, we used Western blot analysis to confirm the expression levels of several pregnancy-related proteins putatively identified as being differentially expressed in somatic cell nuclear transfer (SCNT) vs normal bovine placentae. The expression levels of tissue inhibitor of metalloproteinase-2 (TIMP-2), its downstream protein, matrix metalloproteinase-2 (MMP-2), superoxide dismutase (SOD), vimentin and plasminogen activator inhibitor-1 (PAI) were analysed in the placentae of SCNT cloned Korean native cattle that died immediately after birth and in normal placentae obtained by AI. Our results revealed that TIMP-2 and SOD were up-regulated in SCNT placenta compared with normal placenta, whereas MMP-2 levels were comparable in cloned and normal placentae, and vimentin and PAI were significantly down-regulated in SCNT compared with normal placentae. Our results suggest that key proteins of placental development are abnormally expressed in SCNT cloned bovine placentae, probably resulting in abnormal placental function and clonal mortality.

Production of heterozygous alpha 1,3-galactosyltransferase (GGTA1) knock-out transgenic miniature pigs expressing human CD39

Production of transgenic pigs for use as xenotransplant donors is a solution to the severe shortage of human organs for transplantation. The first barrier to successful xenotransplantation is hyperacute rejection, a rapid, massive humoral immune response directed against the pig carbohydrate GGTA1 epitope. Platelet activation, adherence, and clumping, all major features of thrombotic microangiopathy, are inevitable results of immune-mediated transplant rejection. Human CD39 rapidly hydrolyzes ATP and ADP to AMP; AMP is hydrolyzed by ecto-5′-nucleotidase (CD73) to adenosine, an anti-thrombotic and cardiovascular protective mediator. In this study, we developed a vector-based strategy for ablation of GGTA1 function and concurrent expression of human CD39 (hCD39). An hCD39 expression cassette was constructed to target exon 4 of GGTA1. We established heterozygous GGTA1 knock-out cell lines expressing hCD39 from pig ear fibroblasts for somatic cell nuclear transfer (SCNT). We also described production of heterozygous GGTA1 knock-out piglets expressing hCD39 and analyzed expression and function of the transgene. Human CD39 was expressed in heart, kidney and aorta. Human CD39 knock-in heterozygous ear fibroblast from transgenic cloned pigs, but not in non-transgenic pig’s cells. Expression of GGTA1 gene was lower in the knock-in heterozygous ear fibroblast from transgenic pigs compared to the non-transgenic pig’s cell. The peripheral blood mononuclear cells (PBMC) from the transgenic pigs were more resistant to lysis by pooled complement-preserved normal human serum than that from wild type (WT) pig. Accordingly, GGTA1 mutated piglets expressing hCD39 will provide a new organ source for xenotransplantation research.

Effects of sorbitol on porcine oocyte maturation and embryo development in vitro

In the present study, a porcine system was supplemented with sorbitol during in vitro maturation (IVM) or in vitro culture (IVC), and the effects of sorbitol on oocyte maturation and embryonic development following parthenogenetic activation were assessed. Porcine immature oocytes were treated with different concentrations of sorbitol during IVM, and the resultant metaphase II stage oocytes were activated and cultured in porcine zygote medium-3 (PZM-3) for 7 days. No significant difference was observed in cumulus expansion and the nuclear maturation between the control and sorbitol-treated groups, with the exception of the 100 mM group, which showed significantly decreased nuclear maturation and cumulus expansion. There was no significant difference in the intracellular reactive oxygen species (ROS) levels between oocytes matured with 10 or 20 mM sorbitol and control groups, but 50 and 100 mM groups had significantly higher ROS levels than other groups. The 20 mM group showed significant increases in intracellular glutathione and subsequent blastocyst formation rates following parthenogenetic activation compared with the other groups. During IVC, supplementation with sorbitol significantly reduced blastocyst formation and increased the apoptotic index compared with the control. The apoptotic index of blastocysts from the sorbitol-treated group for entire culture period was significantly higher than those of the partially sorbitol-exposed groups. Based on these findings, it can be concluded that the addition of a low concentration of sorbitol (20 mM) during IVM of porcine oocytes benefits subsequent blastocyst development and improves embryo quality, whereas sorbitol supplement during IVC has a negative effect on blastocyst formation.