This study was designed to determine the effect of electric field strength, duration and fusion buffer in fusion parameters on the rate of membrane fusion between the somatic cell and cytoplast for Korean cattle (HanWoo) somatic cell nuclear transfer (SCNT) procedure. Following electrofusion, effect of 5 or 10 μM Ca2+-ionophore of activation treatment on subsequent development was also evaluated. Cell fusion rates were significantly increased from 23.1% at 20 V/mm to 59.7% at 26 V/mm and 52.9% at 27 V/mm (p<0.05). Due to higher cytoplasmic membrane rupture or cellular lysis, overall efficiency was decreased when the strength was increased to 30 V/mm (18.5%) and 40 V/mm (6.3%) and the fusion rate was also decreased when the strength was at 25 V/mm or below. The optimal duration of electric stimulation was significantly higher in 25 μs than 20 and 30 μs (18.5% versus 9.3% and 6.3%, respectively, p<0.05). Two nonelectrolyte fusion buffers, Zimmermann’s (0.28 M sucrose) and 0.28 M mannitol solution for cell fusion, were used for donor cell and ooplast fusion and the fusion rate was significantly higher in Zimmermann’s cell fusion buffer than in 0.28 M mannitol (91.1% versus 48.4%, respectively, p<0.05). The cleavage and blastocyst formation rates of SCNT bovine embryos activated by 5 μM Ca2+-ionophore was significantly higher than the rates of the embryos activated with 10 μM of Ca2+-ionophore (70.0% versus 42.9% and 22.5% versus 14.3%, respectively; p<0.05). This result is the reverse to that of parthenotes which shows significantly higher cleavage and blastocyst rates in 10 μM Ca2+-ionophore than 5 μM counterpart (65.6% versus 40.3% and 19.5% versus 9.7%, respectively; p<0.05). In conclusion, SCNT couplet fusion by single pulse of 26 V/mm for 25 μs in Zimmermann’s fusion buffer followed by artificial activation with 5 μM Ca2+-ionophore are suggested as optimal fusion and activation methods in Korean cattle SCNT protocol.

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skinderived precursor cells (SKPs) are multipotent, sphereforming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.

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.

Embryonic stem cells have a pluripotency and a potential to differentiate to all type of cells. In our previous study, we have shown that embryonic stem cells (ESCs) lines can be generated from murine parthenogenetic embryos. This parthenogenetic ESCs line can be a useful stem cell source for tissue repair and regeneration. The defect in full-term development of parthenogenetic ESCs line enables researchers to avoid the ethical concerns related with ESCs research. In this study, we presented the results demonstrating that parthenogenetic ESCs can be induced into osteogenic cells by supplementing culture media with ascorbic acid and β-glycerophosphate. These cells showed morphologies of osteogenic cells and it was proven by Von Kossa staining and Alizarin Red staining. Expression of marker genes for osteogenic cells (osteopontin, osteonectin, alkaline phosphatase, osteocalcin, bone-sialoprotein, collagen type1, and Cbfa1) also confirmed osteogenic potential of these cells. These results demonstrate that osteogenic cells can be generated from parthenogenetic ESCs in vitro.

For many years, experience has been accumulated on embryo and gamete manipulation in mammals, The present work is an introduction of these techniques and their possible application in human embryology in s pecific cases, Mammalian c1on ing has been studied by many groups, but the success rate is sti ll low‘ Removal of maternal chromosomes from unfertil ized oocytes and injection of donor cells into enucleated oocytes are the most important factors for the improvement of cloning effi cien cy, Here, we introduce a novel one-step rnicromanipulation (OSM) system and laser-assisted zona pellucida piel'cing technique (LAZP) , 1n genera l, somatic cell nuclear transfer (SCNT) is completed by many processes including enucleation and donor cell fusion , Howevel', OSM is a simple method because donor cell is directly injected into ooplasm without fusion pl'ocess, 1n addition, chromosomal enucleation and donor cell inj ec tion are perfOl‘med simultaneously in OSM, While OSM was a pplied to porcine SCNT, LAZP was a pplied to murine SCNT, This rninirni zed the use of piezo-dri ven micromanipul ator (P1EZO) , I'educing chances 0 1' problems caused by P1EZO pulses, LAZP reduced time that took to pierce zona pellucida in removal of nucleus fl'om oocyte and somatic cell injection, which might have taken longer time with P1EZO, The simple , new OSM and LAZP system may help to enable large scale cloning by reduction of procedural steps, Pa l'thenogenesis de scribes the growth and development of an embryo without fertilization by a male Parthenogenetic ES cell s (PESCs) can be a useful cell source for tissue I'epail‘ and I'egeneration , Moreover , the defects in full-term developrnent of this PESCs enable researc hers to avoid the ethical concern , Here, the author showed that PESCs can differentiate into osteogenic lineage, The PESCs were induced osteogenic dlfferentlatlon The osteoblas t-specific gene expression such as osteocalcine, osteopontine, osteonectin, bone-sialo protein‘ coll agen type-l and alka line phos phatase showed osteogenic potential of differentiated PESCs, The author also focused on the neuronal induction of murine PESCs by simplified neurona l induction system to generate doparninergic (DA) neurons , As a result , PESCs were differentiated into nestin and Tuj-l positive cell s successfully, a lthough t he generation of DA neuron was Illruted For murine embryo cul ture, novel oil-free microtube cul tu re system was applied , This new culture system provides oil-free cu ltu re condi t ions and is easy to handle It was also associated with faster development and mOl'e t l'ophectodel'mal cells , which will enhance the development of murine embl'Yos to fur t hel' stages ,

Somatic cells nuclear transfer (SCNT) is a useful tool in studies of developmental biology and animal cloning. However, SCNT experiments only are allowed to skilled technical experts. In this experiment, laser-assisted zona pellucida piercing tool (LASER) was applied in murine SCNT. LASER minimized the use of piezo-driven micromanipulator (PIEZO), reducing chances of problems caused by PIEZO pulses. LASER reduced time that took to pierce zona pellucida in removal of nucleus from oocyte and somatic cell injection, which might have taken longer time with PIEZO. Time and difficulties that took researcher of equivalent skilled for their experiments were decreased with LASER, and this might affect the improvement of embryonic development. (LASER, 6.2% versus PIEZO, 2.9%; P<0.05). Thus, these data support that the use of LASER can be used for zona pellucida piercing in murine SCNT program as an alternative to PIEZO.

This study was conducted to evaluate the effect of cytochalasin B (CB) treatment in the activation medium on the development of somatic cell nuclear transfer (SCNT) rat embryos. Fetal fibroblast cells were isolated from a Day 14.5 fetus, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM SrCl2 with or without CB for 4 hr, and formation of pseudo-pronucleus (PPN) was checked at 18 hr after activation. Then, they were transferred into day 1 pseudopregnant recipients (Hooded Wistar) or cultured for 5 days to check their developmental competence in vivo or in vitro. The number of PPN was not affected by CB treatment during the activation. However, CB treatment supported pre-implantation development of rat SCNT embryos. Embryos generated by the procedures of SCNT were also capable of implanting, with 1 implantation scar found from a recipient following the transfer of 87 SCNT embryos to four foster mothers. The result of the present study shows that rat SCNT embryo can develop to post-implantation stage following treatment with CB.