The production of transgenic animals using somatic cell nuclear transfer (SCNT) has been widely described. A critical problem in the production of transgenic animals is the uncontrolled constitutive expression of the foreign gene which occasionally results in serious physiological disorders in the transgenic animal. In this study, we designed three different expression vectors that express the hEPO gene. hEPO is a hormone produced by the kidney that promotes the formation of red blood cells by the bone marrow. For the in vitro production of transgenic embryos, the different expression vectors were transduced into holstein ear fibroblast cells, respectively, and GFP expressed donor cells were transferred into enucleated oocytes, and then the reconstructed SCNT embryos were developed into pre-implantation stage. From three replicates, GFP expressed 112 transgenic SCNT embryos were produced. When their cleavage rate and blastocyst rate were compared with non-transgenic SCNT embryos, the results were presented into 73.2% vs. 76.9% and 26.8% vs. 30.6%, respectively, there were no differences. Also, total cell number and ICM cell numbers of day 8 blastocysts were statistically not different between the transgenic SCNT groups (120.6±7.9 and 31.4±8.2) and control SCNT group (128.3±4.8 and 35.3±4.0). The GFP expression levels were presented consecutively high during the culture of transgenic SCNT embryos. By analysis of semi-quantitative RT-PCR, the relative expression levels of hEPO mRNA and pluripotent gene were determined. These results demonstrated that the hEPO expressed transgenic bovine embryos can be efficiently produced in vitro by SCNT technique, while their potential of cloned animal production have to be examined in further study.

Stem cell therapy is undoubtedly the most promising therapeutic approach for neurological disorders. Adipose tissue is ubiquitous and it can be easily harvested in large quantities under local anesthesia with little patient discomfort, making adipose tissue into the ideal large-scale source for research on clinical applications. In this study we monitored the neuronal cell differentiation potential of human adipocyte in the following condition; i) N2 medium containing 200 uM ascorbic acid (AA) and/or 10 uM flavonoid (F) and ⅱ) N2 medium containing AA and/or 10 ng/ml brain derived neurotrophic factor (BDNF) and/or, 200 ng/ml sonic hedgehog (SHH) plus 100 ng/ml fibroblast growth factor (FGF) 8. Adipose stem cells were cultured in above described differentiation condition for three weeks. RT-PCR analysis demonstrated that the mRNA levels of neuronal cell markers in differentiated adipose stem cells. Under the culture condition using N2 medium containing AA, the expression level of nestin (neural progenitor marker) m- RNA was high in all groups, while those of Neuro D, and LEP and FABP4 (adipocyte marker) mRNA were significantly decreased. Also, the addition of BDNF or SHH+FGF8 in N2 medium containing AA enhanced the neural cell differentiation from adipose stem cells, the expression level of Map2 (mature neuron) mRNA was increased, and that of TH (dopaminergic neuron marker) mRNA was high. In addition, we confirmed that the flavonoid addition has effect on the increase of Map2 expression. These results demonstrate that our designed culture condition has effect on the neural cell differentiation of adipose stem cells and this stimulatory effect may be further enhanced by transplantation.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. This study was to examine the in vitro neuron cell differentiation characteristics of our established human (h) iPS cells (IMR90-iPS-1~2) derived from human somatic cells. For the neuron differentiation, well grown hiPS colonies were recovered by collagenase treatment and then suspended cultured in a non-adherent bacteriological culture dish using human embryonic stem (hES) cell culture medium for 4 days. Embryoid bodies were plated and cultured in serum-free ITSFN (insulin/transferrin/selenium/fibronectin) medium for 8 days to select neural precursor cells. Then selected neuronal cells were dissociated, plated onto poly-L-ornithin/laminin coated dish at a concentration of 2 x 105 cells/cm2 and expanded in N2 medium containing 20 ng/ml bFGF, 200 ng/ml SHH and 100 ng/ml FGF-8 for 7 days. For the final differentiation step involved removing agents and culturing for 14 days in 20 ng/ml BDNF added N2 medium. In the neural precursor stage, >90% of nestin positive cells and >50% NCAM positive cells were obtained. Also, in final differentiation step, we confirmed the high percent (>80%) of mature neuron tubulin-β positive cells and approximately >20% of tyrosine hydroxylase positive cells. Also, these results were confirmed by RT-PCR. These results indicated that hiPS cells have potential to generate specific neuron differentiation and especially TH+ neuron was also can be obtained, and thus hiPS-derived neural cells might be an usable source for the study of neuro-degenerative disease.

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures〔10% (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.〕which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into LN2. One-step dilution in straw was done in 25℃ water for 1 min, by placing vertically in the state of plugged- end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.