Porcine blastocyst’s quality derived from in vitro is inferior to in vivo derived blastocysts. In this study, to improve in vitro derived blastocyst’s quality and then establish porcine ESCs (pESCs), we treated in vitro fertilized (IVF) embryos and parthenogenetic activated (PA) embryos with three chemicals: porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), resveratrol (RES) and β-mercaptoethanol (β-ME). The control group was produced using M199 media in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The treatment group is produced using M199 with 2 μM RES in IVM and PZM5 with 10 ng/mL pGM-CSF, 2 μM RES and 10 μM β-ME in IVC. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. In total, 1210 embryos in PA and 612 embryos in IVF evaluated. As results, we observed overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (p<0.05) in the treatment groups (54.5%) compared to the control group (43.4%) in PA and hatched blastocysts rates in day 6 and 7 were also increased significantly. Total cell numbers of blastocyst were significantly higher (p<0.05) in the treatment group (55.1) compared to the control group (45.6). In IVF, hatched blastocysts rates in day 7 were increased significantly, too. After seeding porcine blastocyst, the attachment rates were higher in the treatment group (36.2% in IVF and 32.2% in PA) than the control group (26.6% in IVF and 19.5% in PA). Also, colonization rates and cell line derivation rates were higher in treatment group than control group. Colonization rates of control group were 10.8% in IVF and 2.4% in PA, but treatment group were 17.75% in IVF, and 13.1% in PA. And we investigated the correlation between state of blastocysts and attachment rate. The highest attachment rate is in hatched blastocyst (78.35±15.74 %). So, the novel system increased quality of porcine blastocysts produced from in vitro, subsequently increased attachment rates. The cell line derivation rates were 4.2% (IVF) and 2.4% (PA) in control group. In treatment group, they were 10.0% (IVF) and 7.2% (PA). We established 3 cell lines from PA blastocysts (1 cell line in control group and 2 cell lines in treatment group). All cell line has alkaline phosphatase activity and express pluri-potent markers. In conclusion, the novel system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of porcine blastocysts produced from in vitro, subsequently increased derivation rates of porcine putative ESCs.

Pluripotent stem cells can be derived from both pre- and post-implantation embryos. Embryonic stem cells (ES cells), derived from inner cell mass (ICM) of blastocyst are naïve pluripotent and epiblast stem cells (EpiSCs) derived from post-implantation epiblast are primed pluripotent. The phenotypes and gene expression patterns of the two pluripotent stem cells are different each other and EpiSCs thought to be in a more advanced pluripotent (primed pluripotent state) than mouse ES cells (naïve pluripotent state). Therefore, we questioned whether EpiSCs are less potential to be differentiated into specialized cell types in vitro. EpiSCs were isolated from 5.5～6.5 day post coitum mouse embryos of the post-implantation epiblast. The EpiSCs could differentiate into all tree germ layers in vivo, and expressed pluripotency markers (Oct4, Nanog). Interestingly, EpiSCs also were able to efficiently differentiate into neural stem cells (NSCs). The NSCs differentiated from EpiSCs (EpiSC-NSCs) expressed NSC markers (Nestin, Sox2, and Musasi), self-renewed over passage 20, and could differentiate into two neural subtypes, neurons, astrocytes and oligodendrocytes. Next, we compared global gene expression patterns of EpiSC-NSCs with that of NSCs differentiated from ES cells and brain tissue. Gene expression pattern of brain tissue derived NSCs were closer to ES cell-derived NSCs than EpiSC-NSCs, indicating that the pluripotent stem cell-derived somatic cells could have different characteristics depending on the origin of pluripotent stem cell types. * This work was supported by the Next Generation Bio-Green 21 Program funded by the Rural Development Administration (Grant PJ 008009).

Induced pluripotent stem cells (iPSCs), generated by the overexpression of transcription factors Oct4, Sox2, Klf4 and c‐Myc in somatic cells, are pluripotent. iPSCs reprogrammed from differentiated cells get through a epigenetic modification during reprogramming and finally have the similar epigenetic state to embryonic stem cells (ESCs). In this study, these epigenetic changes were observed in reprogramming of uni‐parental parthenogenetic somatic cells. Furthermore, we have shown that parthenogenetic pattern of imprinted genes were changed during pluripotential reprogramming. Parthenogenetic neural stem cells (pNSCs) containing only maternal alleles regain the biparental imprinting patterns after reprogramming. However, we have yet to define whether the changed imprinted genes are maintained or reverted to the parthenogenetic state when the reprogrammed cells are differentiated into specialized cell types. To address this question, we compared genome‐wide expression profiles of biparental female neural stem cells (fNSCs), parthenogenetic neural stem cells (pNSCs), and NSCs differentiated from parthenogenetic maternal iPSC (miPS‐NSCs). Furthermore, this study establishes the correlation between the alteration of genome methylation and activation of imprinting genes in the parthenogenetic cells and reports for the first time that the silenced PWS‐related imprinted genes are activated in miPS‐NSCs. Our data demonstrated that pluripotential reprogramming of parthenogenetic somatic cells were able to reset the parthenogenetic imprinting patterns; reprogrammed miPSCs showed erasure of maternal methylation imprints and acquisition of methylation in paternally imprinted genes. Furthermore, the changed imprinting patterns were maintained when the reprogrammed cells are differentiated into specialized cell type. * This work was supported by the Next‐Generation BioGreen 21 program (Grant PJ008- 009) funded by the Rural Development Administration, Republic of Korea.

동물의 장기를 인간에게 이식하게 되면 초급성거부반응(Hyperacute rejection, HAR)이 일 어난다. 초급성거부반응은 면역계의 구성요소 중 보체(complement)에 의해 일어나는 거부 반응으로 돼지의 혈관세포 표면에 있는 Galα(1,3)Gal 당분자에 인간의 항체가 즉각 반응하 기 때문에 일어나며, α1,3-galactosyltransferase(α1,3-GT) 유전자는 돼지 혈관세포 표면의 Galα(1,3)Gal 당분자 생성에 관여한다. 따라서 인간에게 돼지의 장기를 이식하기 위해서는 α1,3-galactosyltransferase 유전자를 제거하는 것이 필요한 것으로 알려져 있다. 본 연구 실 의 이전 연구에서, 시카고 미니돼지 귀체세포에서 상동 재조합(Homologous recombination) 을 통해 α1,3-galactosyltransferase 유전자가 제거된 체세포를 개발한 바 있으며, 이 체세 포 를 통하여 α1,3-GT 유전자가 제거된 돼지도 생산된 바 있다. 본 연구에서는, Human serum 처리 시 돼지 세포를 보호해준다고 보고되고 있는 human complement regulator인 human Decay-accelerating factor(hDAF)와 human α1,2-fucosyltransferase(hHT) 유전자를 α1,3 -GT 유전자 위치에 gene targeting하여 동시에 hDAF와 hHT가 발현하는 체세포를 개발하였다. Knock-in vector는 hDAF와 hHT 두 유전자가 발현할 수 있도록 IRES로 연결하였으며 α 1,3-GT 유전자의 start codon을 이용하여 발현할 수 있도록 구축하였다. 구축한 vector는 electroporation을 통해 미니돼지 체세포에 도입하였으며, PCR 결과 α1,3-GT 유전자 위치 에 서 상동 재조합이 일어났음을 확인하였다. Positive-negative 선별 방법을 통해 얻은 gene targeting된 체세포는 RT-PCR에 의해 hDAF와 hHT 유전자의 발현이 확인되었으며, 대조군 (NIH minipig)에 비해 α1,3-GT 유전자의 발현이 감소하였다. 또한, 이들 세포에 100% human complement serum을 처리하였을 때 Knock-in 세포가 대조군에 비해 30% 정도 더 높 은 생존율을 보였다. 따라서 개발된 체세포는 이종간 장기이식을 위한 돼지 생산과 함께 이를 이용한 이종간의 장기 이식 시 초급성 거부반응을 억제하는 데 사용 될 수 있을 것으로 생각된다.

Hyperacture rejection (HAR) of pig organs, upon xenotransplantation into primates, could partly be overcome by knocking out the alpha-Gal gene. However, xenotransplanted organs may still undergo immunological acture rejection (AR) or acute vascular rejection (AVR). Among several genes involved in AR and AVR, the hCD47 evades the monocyte/ macrophage mediated phagocytosis by identifying the self/non-self signal (CD47-SIRPa) whereas hTFPI participates in the regulation of coagulation pathway by acting upstream of the thrombin. In this study, we investigated hCD47 and hTFPI as two possible candidates for avoiding AR and AVR, respectively upon pig-to-human xenotransplantation. A co-expression vector for hCD47 and hTFPI was constructed using 2A peptides system (F2A) and transfected into the porcine kidney cell line (PK-15). The transfected cells stably expressed both hCD47 and hTFPI mRNA and proteins. Co-culture of non-transfected, hCD47-transfected, hTFPI-transfected or hCD47+hTPFI-transfected PK15 cells with natural killer (NK) cells, monocytes and macrophages confirmed the cytotoxic effect of hCD47 and revealed a synergistic effect of hCD47 and hTFPI co-transfection. There was an attractive survivability of 25～30% on each type of innate immune cell, NK cell and macrophage. These results suggest that transgenic pigs, genetically modified for hCD47 and hTFPI may be useful for overcoming xenograft rejection. Furthermore, cotransfection with hTFPI may enhance the cytotoxic effect of hCD47, possibly by assisting the hCD47-SIRPa binding by an unknown mechanism.

Among laboratory animals, pigs are anatomically and physiologically closer to human. Transgenic (TG) pigs can be widely applied as models of human diseases. Many researchers created TG pigs which have specific modified genome under a constitutively active promoter. A constitutively active promoter is effective to express a target gene, but the uncontrollable expression often results in unwanted outcomes. In this study, as a way to solve these problems, we tried to regulate the expression of target genes by tetracycline (Tet) on/off system. We tested the operation of Tet on/off system in TG donor cells. Miniature porcine fetal fibroblasts were transfected with universal doxycycline- inducible vector and an enhanced green fluorescent protein (eGFP) was used as the target gene. The induced transgene expression by doxycycline was detected on fluorescence microscopy. On one day after 1 μg/ml doxycycline treatment, the fluorescence intensity for TG cells was increased. And we then performed Somatic Cell Nuclear Transfer (SCNT) to confirm the working of Tet on/off system in the porcine SCNT-TG embryos. Total 649 transgenic porcine SCNT embryos were made. From these, 64 of SCNT embryos used in invitroculturewith1 μg/mldoxycycline. Among these porcine SCNT-TG embryos, 39 embryos (60.9%) were cleaved. Finally, 15 transgenic porcine SCNT embryos developed blastocyst. Induced transgene expression was observed all of cleaved embryos and blastocysts. The remaining 585 embryos were transferred to 6 surrogates. On 25 days after embryo transfer, two surrogates were diagnosed as pregnant (pregnancy rate =33.3%). On day 113 (one day prior to delivery), we obtained six cloned TG piglets from first pregnant surrogate. Unfortunately, all TG piglets died because their surrogate died suddenly at delivery time. However, we could obtain the TG cell lines from the cloned TG piglets. Being analyzed by PCR, all piglets were found to be eGFP gene targeted. Now, second pregnant surrogate have maintained at 80 days after embryo transfer and shown more than three embryonic sacs. This data suggested that, Tet on/off system can control target gene expression in transgenic porcine SCNT embryos. This result has presented new possibilities of regulation of target gene expression in cloned TG pigs by Tet on/off system. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

The influenza viruses can be spread from birds to people. In this process, the pig is the intermediate host, and this virus is amplified and produces many mutations in pigs. Therefore, we attempted to develop the influenza-resistant pigs for the study of the virulence test and the transgenic (TG) animal model for translational research. At interferon- α, γ treated cells, the porcine Mx2 protein has been observed near the nuclear envelope and inhibits influenza virus proliferation, but not in common cells. So, we tried to produce the Mx2 gene over-expressed pig by somatic cell nuclear transfer(SCNT).First, we establish the Mx2 gene over expressed cells for the preparation of the TG donor cells. Porcine fetal fibroblasts were transfected with cytomegalo virus vector which include the porcine Mx2 gene. The established transgenic cell was injected into the enucleated ooplasm for the production of the Mx2-TG cloned embryos. Total, 511 female TG porcine SCNT embryos (TG-SCNTembryos) were made. The 511 female TG-SCNT embryos were transferred to five surrogates. On 25 days after embryo transfer, two of female embryos’ surrogates were diagnosed as pregnant (pregnancy rate, 40%). On day 114, we obtained six cloned piglets and four mummies from two of female embryos’ surrogate. Being analyzed by PCR, all female piglets were not integrated with Mx2 gene. Hereby, we again established newly male MX-TG cell line for donor cell of SCNT. 427 male TG-SCNT embryos were made. From these, 38 of male TG-SCNT embryos were cultured in in vitro to confirm the developmental capacity of TG-SCNT embryos. Among these porcine SCNT-TG embryos, 26 embryos (68.4%) were cleaved. Finally, 5 transgenic porcine SCNT embryos (13.2%) developed to the blastocyst stage. All male TGSCNT blastocysts were proved to be integrated with Mx2 gene as PCR analysis. Therefore, we expect that newly birth male piglets will be targeted with MX2 gene. The remaining 389 male embryos were transferred to four surrogates. On 25 days after embryo transfer, one of male embryos’ surrogates was diagnosed as pregnant (pregnancy rate, 25%). Now, pregnant surrogate have maintained at 88 days after embryo transfer and shown more than eight embryonic sacs. This study has presented new possibilities of production of influenza virus resistant pig by SCNT for translational research. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

The T-cell receptor (TCR) engages with an antigen and initiates a signaling cascade that leads to the activation of transcription factors. Roquin, a protein encoded by the RC- 3H1 gene and characterized as an immune regulator, was recently identified as a novel RING-type ubiquitin ligase family member, but the mechanisms by which Roquin regulates T-cell responses are unclear. We used the EL-4 murine lymphoma cell line to elucidate the role of Roquin in vitro. Roquin-overexpressing EL-4 cells became hyper-responsive after anti-CD3/CD28 stimulation in vitro and were a major source of the cytokines IL-2 and TNF-α. Upon activation, these cells showed particularly enhanced production of IL-2 and TNF-α. To clarify the important role played by Roquin in T-cell responses ex vivo, we generated T-cell-specific Roquin transgenic (Tg) mice. Roquin-Tg CD4+ T-cells showed enhanced production of IL-2 and TNF-α in response to TCR stimulation with anti-CD28 co-stimulation. Further studies are necessary to investigate the role of Roquin in the regulation of primary T-cell activation, survival, and differentiation.

본 연구는 핵산가수분해 기능이 있는 항체인 3D8 scFv(single-chain variable fragment) 유전자를 형질전환 닭에서 발현시키고 3D8 scFv 항체의 항-바이러스 기능을 검증하고자 한다. 연구는 in vitro(형질전환 닭유래 세포)와 in vivo(형질전환 닭)로 나누어서 수행하고자 한다. 먼저 닭 태아섬유아세포는 형질전환 닭과 일반 닭을 각각 인공수정을 시킨 후 생산된 10일차 수정란의 태아에서 CEF(Chicken Embryo Fibroblast)를 수집하였다. 3D8 scFv 유전 자가 삽입된 CEF 세포는 항생제(puromycin)를 이용하여 형질전환 세포만을 선발하였다. 그 리고 선발된 CEF 세포는 면역염색을 이용하여 3D8 scFv 유전자가 세포내에서 단백질로 발현됨을 확인하였다. 그리고 선발된 세포들의 항-바이러스 기능 검증은 GFP 유전자로 표 지된 재조합 NDV(Newcastle Disease Virus)를 세포에 직접 감염시키고, 세포내에서 발현 하 는 GFP의 발현량을 FACS를 이용하여 정량하는 방법으로 항-바이러스 기능을 조사하였다. 이들 가운데 항-바이러스 기능이 있을 것으로 예상되는 형질전환 2계통에 대하여 항-바이러스 기능을 검증을 준비하고 있다.

Genetic modification of the pig of which the gene is relevant to human diseases allows the pig to be used as a source of biomedical animal model. The promoter which could drive efficient expression constitutively or specifically of the interest gene in porcine organs is essentially required to increase versatility of a biomedical porcine model. In this study, we compared different promoters of activities driving efficient expression in different types of porcine cells including primary fibroblasts, kidney-derived PK-15, and primary endothelial cells (EC). To this end, we inserted CMV, EF1-α, CMV/EF1-α, CAG, human and porcine membrane cofactor protein gene promoters(MCP and Mcp), and porcine intercellular adhesion molecule-2 (Icam-2) promoter into pGL3 basic vector. Luciferase assay revealed that CAG promoter led to highest promoter activity in fibroblasts and PK-15 cells. CMV, EF1-α, CMV/EF1-α promoters showed moderate activities for luciferase expression in fibroblasts and PK-15 cells. Interestingly, CMV/EF1-α promoter, in which CMV promoter was linked to the front of EF1-α promoter as an enhancer led to highest luciferase expression in EC. The MCP, Mcp and Icam-2 promoters showed very low level of luciferase expression in three types of cells. Taken together, this study demonstrated that promoter activity in different porcine cells is differently expressed.

혈액응고 제 8인자(FVIII)는 혈액 내 존재하는 당단백질의 하나로, 혈액응고의 내인성 기 작에 기능한다. 이러한 FVIII의 결핍은 A형 혈우병을 야기하는 것으로 알려져 있으며, A형 혈우병 환자는 FVIII의 지속적인 주사에 의해 치료되고 있다. 본 연구는 A형 혈우병의 치료 제로써 활용 가능한 B-domain이 변형된 재조합 인간 혈액응고 제 8인자 (dB747)를 유즙으 로 분비하는 형질전환 돼지의 유즙으로부터 크로마토그래피적인 방법으로 dB747의 효율 적 인 정제를 위한 방법을 제공하기 위하여 수행되었다. 연구는 dB747을 유즙으로 발현하는 형질전환 돼지의 유즙을 착유하여 원심분리를 통해 지질과 불순물을 제거하고, 유즙 내 복 합적으로 존재하는 수 많은 단백질을 분획하기 위한 전처리 과정으로써 일반적으로 유즙 단백질을 침전시킨다고 알려진 zinc chloride, calcium chloride와 일반적인 단백질 침전에 널리 이용되는 ammonium sulfate를 농도 별로 처리하여 분획의 정도를 확인하였다. 전처 리 과정을 통해 분획된 유즙을 이용하여 음이온 교환 크로마토그래피, 친화 크로마토그래피 방 법으로 정제하였다. 음이온 교환 크로마토그래피는 혈장 유래의 FVIII을 정제하는데 유리하 다고 알려진 Q sepharose FF 컬럼을 이용하여 대표적인 두 가지 sodium acetate와 sodium citrate buffer의 효과를 비교하였다. 또한, 친화 크로마토그래피는 B-domain이 결여된 재조합 인간 혈액응고 제 8인자(BDDrFVIII)을 정제하는데 이용 가능하다고 알려진 펩타이드 TN8.2와 EYHSWEYC를 리간드로 사용한 컬럼을 제작하여 수행하였다. 그 결과, dB747이 포함된 형질전환 돼지의 유즙을 분획하기 위해서는 ammonium sulfate의 연속적인 처리를 통해 포화도 30%의 상층액을 회수하여 포화도 50%가 되도록 ammonium sulfate를 첨가하 였을 때 생성되는 pellet을 이용하는 것이 가장 효율적인 것으로 나타났다. 효율적인 전처리 과정을 거친 유즙을 이용한 크로마토그래피 결과, 혈장 유래의 FVIII의 정제에 대한 이전의 보고와는 다르게 dB747의 회수율과 순도가 낮았다. EYHSWEYC를 이용한 친화 크로마토 그래피의 경우, 알려진 것과는 다르게 dB747과 결합하지 않는 것으로 확인되었으며, TN8.2 를 이용한 친화 크로마토그래피 역시 dB747를 정제하는데 이용하기 어려웠다. 또한, 크로 마토그래피 용출액 내에 20～40 kDa의 단백질이 나타나는 것을 확인하였으며, 이는 문헌 조사를 통해 카제인으로 유추할 수 있었다. 이러한 결과는 dB747이 B-domain 영역이 변형 되는 과정에서 기존의 FVIII 또는 BDDrFVIII과 단백질의 성질이 달라짐으로써 기존에 알려 진 정제 조건이 알맞지 않기 때문에 회수율과 순도가 낮았던 것으로 사료된다. 따라서 dB747의 성질을 이해하고 최적화된 크로마토그래피 조건을 확립하기 위한 연구가 필요할 것이다. 또한, 카제인과 같은 유즙 단백질이 크로마토그래피 용출액에서 나타나는 것으로 보아, 유즙 단백질은 음이온 교환 크로마토그래피나 친화 크로마토그래피에 의해 제거되지 않음을 확인할 수 있었다. 그러므로 초기 전처리 단계에서 유즙 단백질을 제거하기 위한 방 법의 연구가 유즙 내 목적 단백질을 정제하는데 있어서 중요한 문제가 될 것이다.

In this study, we examined whether Hanganutziu‐Deicher (H‐D) antigens are important as an immunogenic non‐a1,3‐galactose (Gal) epitope in pigs with a disrupted a1,3‐ galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells. A total of 1800 somatic cell nuclear transfer (SCNT) embryos were transferred to 10 recipients. One recipient developed to term and naturally delivered two piglets. The a1,3‐galactosyltransferase activity in lung, liver, spleen, and testis of heterozygote a1,3‐galactosyltransferase gene knockout (GalT‐KO) pigs was significantly decreased, whereas brain and heart showed very low decreasing levels of a1,3‐ galactosyltransferase activity when compared to those of control. Enzyme‐linked lectinosorbent assay showed that the heterozygote GalT‐KO pig had more sialyla2,6‐ and sialyla2,3‐ linked glycan than the control. Furthermore, the heart, liver, and kidney of the heterozygote GalT‐KO pig had a higher N‐glycolylneuraminic acid (Neu5Gc) content than the control, whereas the lung of the heterozygote GalT‐KO pig had Neu5Gc content similar to the control. Collectively, the data strongly indicated that Neu5Gc is a more critical xenoantigen to overcoming the next acute immune rejection in pig to human xenotransplantation.

Calcineurin-binding protein 1 (Cabin1) regulates calcineurin phosphatase activity as well as the activation, apoptosis, and inflammatory responses of fibroblast-like synoviocytes (FLSs), which actively participate in the chronic inflammatory responses in rheumatoid arthritis (RA). However, the mechanism of action of Cabin1 in FLS apoptosis is not clear. The aim of this study was to define the regulatory role of Cabin1 in FLSs of mice with collagen-induced arthritis (CIA). Transgenic mice overexpressing human Cabin1 in joint tissues, under the control of a type II collagen promoter, were generated. hCabin1 expression in joints and FLSs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. The expression of cytokines, matrix metalloproteinases (MMPs), and apoptosis-related genes in FLSs was determined by enzyme- linked immunosorbent assay, gelatin zymography, and RT-PCR, respectively. Joints were histologically examined after H&E and TRAP staining. hCabin1-transgenic CIA mice had less severe arthritis than wild-type CIA mice, based on hind paw thickness and histology. This was accompanied by significantly enhanced apoptosis in transgenic mice, evidenced by significantly more TUNEL-positive cells in synovial tissues. The expression of inflammatory cytokines and MMPs was reduced, and the transgenic CIA mice exhibited decreased Akt activation and increased expression of p53, caspase-3, caspase-9, and Bax. hCabin1 plays a critical role in promoting apoptosis of FLSs and in attenuating inflammation and the destruction of cartilage and bone in RA. These findings help elucidate the pathogenic mechanisms of RA and suggest that Cabin1 is a potential target for RA treatment.