Background: Canine induced pluripotent stem cells (iPSCs) are an attractive source for veterinary regenerative medicine, disease modeling, and drug development. Here we used vitamin C (Vc) to improve the reprogramming efficiency of canine iPSCs, and its functions in the reprogramming process were elucidated. Methods: Retroviral transduction of Oct4, Sox2, Klf4, c-Myc (OSKM), and GFP was employed to induce reprogramming in canine fetal fibroblasts. Following transduction, the culture medium was subsequently replaced with ESC medium containing Vc to determine the effect on reprogramming activity. Results: The number of AP-positive iPSC colonies dramatically increased in culture conditions supplemented with Vc. Vc enhanced the efficacy of retrovirus transduction, which appears to be correlated with enhanced cell proliferation capacity. To confirm the characteristics of the Vc-treated iPSCs, the cells were cultured to passage 5, and pluripotency markers including Oct4, Sox2, Nanog, and Tra-1-60 were observed by immunocytochemistry. The expression of endogenous pluripotent genes (Oct4, Nanog, Rex1, and telomerase) were also verified by PCR. The complete silencing of exogenously transduced human OSKM factors was observed exclusively in canine iPSCs treated with Vc. Canine iPSCs treated with Vc are capable of forming embryoid bodies in vitro and have spontaneously differentiated into three germ layers. Conclusions: Our findings emphasize a straightforward method for enhancing the efficiency of canine iPSC generation and provide insight into the Vc effect on the reprogramming process.

Somatic cell nuclear transfer (SCNT) in pigs has been used as a very important tool to produce transgenic for the pharmaceutical protein, xenotransplantation, and disease model and basic research of cloned animals. However, the production efficiency of SCNT embryos is very low in pigs and miniature pigs. The type of donor cell is an important factor influencing the production efficiency of these cloned pigs. Here, we investigated the developmental efficiency of SCNT embryos to blastocysts and full term development using fetal fibroblasts (FF) and mesenchymal stem cells (MSCs) to identify a suitable cell type as donor cell. We isolated each MSCs and FF from the femoral region and fetus. Cultured donor cell was injected into matured embryos for cloning. After that, we transferred cloned embryos into surrogate mothers. In term of in vitro development, the SCNT embryos that used MSCs had significantly higher in cleavage rates than those of FF (81.5% vs. 72%) (p<0.05), but the blastocyst formation rates and apoptotic cell ratio was similar (15.1%, 6.18% vs. 20.8%, 9.32%). After embryo transferred to surrogates, nine and nineteen clone piglets were obtained from the MSCs and FF group, respectively, without significant differences in pregnancy and birth rate (50%, 40% vs. 52.3%, 45.4%) (p>0.05). Moreover, there was no significant difference in the corpus hemorrhagicum numbers of ovary, according to pregnancy, abortion, and delivery of surrogate mothers between MSCs and FF groups. Therefore, the MSCs and FF are useful donor cells for production of clone piglets through SCNT, and can be used as important basic data for improving the efficiency of production of transgenic clone pigs in the future.

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of IFN-γ. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

5‐aza‐2’‐deoxyctidine (5‐aza‐dC) is DNA methylation inhibitor and Trichostatin A (TSA) is histone deacytlase inhibitor, both of them can alter the level of the epigenetic modification of cells. The objective of this study was to investigate the effects of treatment with 5‐aza‐dC and TSA into fetal fibroblasts on the development of porcine nuclear transfer (NT) embryos. In this study, experiments were performed in order to modify epigenetic status in donor cells and evaluate developmental potential of NT embryos. 5‐ aza‐dC or TSA or combining treatment of TSA and 5‐aza‐dC was treated into growing donor cells for 1 h exposure and development of NT embryos was evaluated. Experiment was performed with 3 groups: control group (donor cells without treatment); TSA group (donor cell treated with 50 nM TSA for 1 h); TSA + 5‐aza‐dC group (donor cells were treated with 50 nM TSA and 5 nM 5‐aza‐dC for 1 h); TSA+1/2(5‐aza‐dC) group (donor cells were treated with 50 nM TSA for 1h and subsequently treated with 2.5 nM 5‐aza‐dC for another 1h). When donor cells were individually treated with 5 nM 5‐aza‐dC or 50 nM TSA for 1h, the blastocyst rate of NT embryos increased significantly compared with control group [18.8% vs 13.4% (5 nM 5‐aza‐dC group vs control group), and 26.2% vs 11.8% (50 nM TSA group vs control group), p<0.05]. However, the blastocyst rate in combining treatment group (50 nM TSA + 5 nM 5‐aza‐dC) did not increase compare with control group (12.3% vs 11.8%, p>0.05). When the donor cell were individually treated with 50nM TSA for 1 h firstly and then treated with 2.5 nM 5‐aza‐dC for another 1h, the blastocyst rate was significantly improved compared with control and TSA group (28% vs 10.2% and 23.7%, p<0.05). The present study suggested that donor cells treated with TSA or low concentration of TSA+5‐azadC in short time exposure may enhance the development of porcine NT embryo.

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells (1.09 × 105 eell₃/ml). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR:64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% (30 × 104 eell₃/ml) or 20% KSR (4.8 × 104 cells/ml) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.

체세포의 배양 방법은 체세포 핵이식에 의한 형질 전환 돼지 생산에 있어서 중요한 요인 중 하나이다. 본 연구에서는 돼지 태아 섬유 아세포의 효율적인 배양 방법을 수려하였다. 돼지 태아 섬유 아세포는 임신 33일째 태아로부터 제조하였으며, 돼지 태아 섬유아세포의 증식을 혈청과 배지 종류별로 분석하였다. 그 결과, 15% ES screened FBS가 포함된 DMEM 배지에서의 배양은 15% FBS보다 세포수의 증가가 훨씬 더 빠르게 나타났다. 또한, 태아

본 연구의 목적은 요를 통해 hFSH를 발현하는 형질 전환 소의 생산이다. 요의 분비와 관련 있는 유전자로서 mUII promoter를 사용하여 hFSH유전자를 구성했다. 태아섬유아세포(KbFF)는 임신 45일령의 태아(male)에서 채취하였다. hFSH gene은 pcDNA3(neo) vector와 같이 KbFF 세포에 electroporation 방법으로 transfection하였다. 유전자를 transfection한 세포는 G-418로 2주 동안

본 연구는 한우 성체 유래 귀세포(Korean bovine ear skin fibroblasts, KbESF)와 태아 섬유아세포(Korean bovine fetal fibroblasts, KbFF)를 이용한 체세포 복제(SCNT) 시 세포종류, 배양기간 그리고 융합방법이 핵이식 수정란의 발달에 미치는 영향을 알아보기 위하여 실시하였다. 태아 섬유아세포는 임신 51일령의 한우태아에서 분리하였고, 귀세포는 28개월령의 성우의 귀에서 채취하였다. 세포는 15주 동안 체외에서 배양하며 체세포 핵이식(SCNT)에 공시하였다. 융합방법을 비교하기 위해 챔버방법과 전극 바늘을 이용한 방법으로 핵과 세포질을 융합하였다. 세포의 doubling time은 KbFF에서 17.3시간, KbESF에서 24.3시간으로 나타났다. 핵이식 후 융합과 분할율은 needle 방법에서 보다 유의적으로 높았으나(각 각 76.1과 81.2%, P<0.05), 배반포 발달율은 차이가 없었다. KbESF의 경우, 배반포 발달율은 passage 5~9(39.4%)와 13~15(40.4%)에서 passage 1~4에 비하여 유의적으로 높았다(P<0.05). KbFF의 경우, 융합율은 passage 5~8과 13~15에서 각각 75.0 및 76.8%로 passage 1~4(61.5%)보다 높았으나, 난분할율과 배반포 발달율은 차이가 없었다. 결론적으로, SCNT 수정란의 발달은 융합 방법에 의해 영향을 받을 수 있으나, 계대배양 15회까지 장기배양을 한 경우는 복제수정란의 발육에 영향을 주지 않는 것으로 판단된다.