Somatic cell nuclear transfer (SCNT) technique is a key point of producing transgenic animal disease models. During in vitro production of SCNT embryo, the quality of matured oocytes are one of the important factors that regulate embryo developmental capacity. In preliminary test, we confirmed the effect of fibroblast growth factor 10 (FGF10) on porcine oocyte maturation. In this study, we investigated the developmental potential of SCNT embryos treated with the 10 ng/ml FGF10 (10 F) during in vitro maturation of recipient oocytes. The polar body emission rate was significantly higher in the 10 F treated group than control group. After SCNT, although the rate of fusion was no significant difference, the rate of cleavage and blastocyst formation was significantly increased in the 10 F treated group (p<0.05). In 10 F treated group, the total cell number was increased and the percentage of apoptotic cell was decreased in the blastocyst stage at day 7 (p<0.1). The transcription level of apoptosis relative gene, Casp3 was significantly decreased, while anti-apoptosis gene BCL2l1 was increased in the 10 F treated group compared to control group. The 10 F treated group was highly expressed the reprogramming related genes, Sox2 and POU5f1. Also, the first cleaving time was more faster and the percentage of cell block was significantly lower in 10 F treated group than in control group. In this study, we confirmed that 10 ng/ml FGF10 has effect on enhance the oocyte maturation and developmental capacity. These results demonstrate that FGF10 treatment can be used for in vitro development of porcine SCNT embryos and subsequent production of transgenic animal model.

Gene targeting is a genetic technique that utilizes homologous recombination between an engineered exogenous DNA fragments with the endogenous genome of an organism. In domestic animal, gene targeting has provided an important tool for producing Knock-out pig for GGTA1 gene to use xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse ES cells. So the application of gene targeting in somatic cells has been limited by its low efficiency. Recently, knock-out rat and mouse was generated by introducing nonhomologous end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knock-out and knock-in techniques in domestic animal is very important in biomedical research. In this present study, we investigated whether homologous recombination events occurs at cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. CMAH-targeted ZFN DNA and mRNA were purchased from SIGMA-Aldrich. CMAH neo targeting vector consists of the neomycin resistance gene as a positive selectable marker gene, 789 bp 5’ arm and 763 bp 3’ arm from Exon 8 of CMAH gene. For transfection, the targeting vector and ZFN DNA or mRNA were introduced into ear fibroblasts cells of Chicago miniature pig by electroporation. After selection of G-418, PCR analysis was performed using 213 colonies transfected with ZFN DNA or mRNA. As a result, 39 positive colonies were identified in colonies transfected with ZFN DNA or mRNA. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in the production of CMAH knock-out for xenotransplantation.

Several human leukocyte subsets including natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and polymorphonuclear neutrophils (PMN) participate in cellular immune responses directed against vascularized pig-to-human xenografts. As these leukocytes express the death receptor Fas either constitutively (PMN) or upon activation (NK, CTL), we explored in vitro whether the transgenic expression of membrane-bound human Fas ligand (mFasL) on porcine fetal fibroblasts is a valuable strategy to protect porcine xenografts. cDNA of mFasL carrying the deletion at the cleavage site with metalloproteinase and lacking the death domain in its cytoplasmic tail was subcloned into pCAGGS expression vector driven by the chicken β-actin promoter containing blastidin- resistance cassette. The mFasL expression vector was transfected into mini-pig fetal fibroblasts by lipofection method. Blastidin-resistant cells were screened by PCR and FISH. The expression of mFasL was confirmed by Western blot and FACS with the mouse anti-human FasL antibody. Interaction of two transgenic clonal cell lines with human leukocytes was analyzed using functional assay for cytotoxicity. mFasL expressed on porcine fetal fibroblasts protected porcine fetal fibroblasts against killing mediated by human NK cells. The rate of NK cell mediated cytotoxicity was significantly reduced in transgenic clonal cells (54±10.80%) compared to normal minipig fetal fibroblasts. This result indicated that grafts of transgenic pigs expressing mFasL could control the cellular immune response to xenografts, and create a window of opportunity to facilitate xenograft survival.

To examine the differential expression of proteins during the cycling (70～80% confluences) and G0/G1 (full confluences) phases in porcine fetal fibroblast cells, we used a global proteomics approach by 2‐D gel electrophoresis (2‐DE) and MALDI‐TOF‐MS. Cycling cell were harvested at approximately 70% to 80% confluent state while cells in G0/G1 phase were recovered after maintenance of a confluent state for 48 hr. Cellular proteins with isoelectric points ranging between 3.0～10.0, were analyzed by 2‐DE with 2 replicates of each sample. A total of approximately 700 spots were detected by 2‐D gels stained with Coomassie brilliant blue. On comparing the cell samples obtained from the cycling and G0/G1 phases, a total of 13 spots were identified as differentially expressed proteins, of which 8 spots were up‐regulated in the cycling cell and 5 were up‐regulated in the G0/G1 phase. Differentially expressed proteins included K3 keratin, similar to serine protease 23 precursor, protein disulfide‐isomerase A3, microsomal protease ER‐60, alpha‐actinin‐2, and heat‐shock protein 90 beta. The identified proteins were grouped on the basis of their basic functions such as molecular binding, catabolic, cell growth, and transcription regulatory proteins. Our results show expression profiles of key proteins in porcine fetal fibroblast cells during different cell cycle status.

This study was conducted to investigate the efficacy of vitrification procedure for the cryopreservation of porcine oocytes and the utilization of vitrified oocytes as recipient cytoplasts for somatic cell nuclear transfer (NT), and observed that porcine oocytes are evaluated by pronuclear formation, and parthenogenetic development. Single fetal donor cells were deposited into the perivitelline space of vitrified enucleation oocytes, followed by electrical fusion and activation. NT embryos were cultured in NCSU-23 medium supplemented with 5% FBS, at in 5% and air. 1. When the developmental rates of the oocytes after being culture for hours vitrified with EDS and ETS were 42.0%, 38.0%, respectively. This results were lower than the control group(62.2%). 2. When the developmental rates of the oocytes after being culture for hours vitrified-thawed with sucrose and glucose, 5% PVP, NCSU-23 supplemented with 10% FBS were 33.3%, 25.9%, respectively. This results were lower than the control group(55.6%). 3. The fusion and development to the blastocyst stage between the NT embryos constructed with the vitrified and non-vitrified oocytes were significant differences. Developmental rate of oocytes and NT embryos constructed with the vitrified or non-vitrified oocytes were , respectively.

본 연구는 돼지 난포란의 동결보존 후 생존성과 난자의 활성화 처리에 따른 체외발생율과 이를 이용한 핵 이식배의 체외발생율을 조사하였다. 활성화 처리된 배는 FBS가 첨가된 NCSU 23 배양액으로 , 와 air의 조건으로 배양하였다. 1. 난포란을 EDS와 PVP로 동결 후 FBS가 첨가된 NCSU 23 배양액으로 시간 배양했을 때 체외발생율은 로서 대조군인 비동결 난포란의 체외발생율 에 비해 낮았다. 2. Ethanol과 cyclojexamide로 처

본 연구에서는 clonal cell lines을 효율적으로 확립할 수 있는 방법을 제시하기 위하여 배양액 내에 catalase와 ME 첨가가 clonal cell line 확립 효율에 미치는 영향을 검토하였다. 임신 50일령의 암퇘지에서 얻은 태아섬유아세포를 2회 passage한 후 동결 보관하였다가 실험에 이용하였다. 단일세포를 catalase나 ME가 첨가된 배양액이 들어 있는 96-well dish로 옮겨 배양하였다. 단층이 형성된 세포는 4-we

본 연구는 돼지 태아 섬유아세포유래 공여세포를 미세주입에 의해 주입 후 재 조합한 핵 이식 배에 대한 배양액, 세포주기의 동기화, 배양시간 및 난자의 활성화에 따른 융합율과 체외발생율에 대해 조사하였다. 핵 이식 배를 NCSU-23, TL Hepes 및 TZM-3 배양액으로 1시간 및 8시간 배양하였을 때 배반포로의 분할율은 각각 15.6%, 14.0%, 15.0% 및 13.9%, 10.5%, 13.3%로서 배양액 및 시간에 따른 분할율의 유의적인 차이