Abnormal epigenetic reprogramming of donor nuclei is supposed to be one of the factors that causes low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone acetylase, and so development of SCNT embryos could be increased by treatment with TSA. In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from NT (nuclear transfer) by investigating the status of histone acetylation in TSA-treated and control NT embryos and the expression of developmental related genes. In this study, we found that incubating　NT embryos with 40nM TSA for 24h after activation could improved the blastocyst formation rate from 13.7% to 32.5%. Thechange in histone acetylation level as a reslut of TSA treatment were validated using immunofluorescence and confocal microscopy. Immunofluorescence results indicated that the level of aetylation at histone 3 lysine 18 (AcH3K18) was increased at early embryo development stage after TSA treatment. furthermore, we compared the expression patterns of several genes (developmental related genes; Oct4, Sox2, Nanog, Cdx2, the imprinting genes; igf2r). TSA treatment improved the expression of development related genes such as Oct4, Cdx2, Nanog as well as the imprinted genes like igf2r. In conclusion, our results demonstrated that TSA treatment improves the in vitro development of porcine NT embryos, increased the global histone acetylation (AcH3K18) and enhances the expression of some developmentally important genes (Oct4, Cdx2, Nanog) at blastocyst stages.

국내산 녹두전분과 도토리전분을 이용하여 초산 녹두 및 초산 도토리전분을 제조하고, 이화학적 특성 및 겔의 조직감을 비교하였다. 무수 초산 6%를 반응시켜 제조한 초산 녹두와 초산 도토리전분의 %acetyl 함량은 1.88%과 1.53% 였고, 치환도는 0.07과 0.06이었다. 전분의 색도를 측정한 결과, L값과 a값은 증가하였고, b값은 감소하여 초산 처리가 전분의 색에도 영향을 주었음을 알 수 있었다. 또한 초 산전분은 전분 chain 내부에 초산기가 도입되어 원료 전분 에 비해 blue value, 팽윤력 및 용해도가 증가하는 것을 알 수 있었다. 신속호화점도계(RVA)를 이용하여 측정한 호화 특성은 녹두전분과 도토리전분의 호화온도가 각각 70.4oC, 75.7oC로 나타났고, 초산 녹두 및 초산 도토리전분은 각각64.4oC, 74.8oC로 나타나 초산 처리 시 호화온도가 감소하는 것을 알 수 있었으나, 도토리전분의 경우 초산 도토리 전분과의 유의적인 차이는 나타나지 않았다. 초산전분은 원료 전분과 최고점도, setback, breakdown에도 차이를 보였는데, 초산 녹두전분은 최고점도 164.9 RVU, setback 70.9 및 breakdown 53.4로 녹두전분의 최고점도 195.0 RVU, setback 110.6 및 breakdown 69.0에 비해 감소하였고, 초산 도토리전분은 최고점도 212.1 RVU, setback 128.7, 및 breakdown 50.3으로 도토리전분의 최고점도 198.3 RVU, setback 87.0 및 breakdown 38.2보다 증가하는 특성을 나타내었다. 초산전분으로 겔을 제조하여 조직감을 측정한 결과 초산 녹두전분은 겔을 형성하지 못하였고, 초산 도토리전분은 원료 전분 겔에 비해 경도와 탄성이 감소하였으며, 응집성, 검성, 씹힘성이 증가하여 겔이 무른 특성을 나타내었다.

Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).

In this study, we produced the recombinant lunasin peptide using E. coli and P. pastoris, and evaluated biological activity of the recombinant lunasin peptide. Lunasin peptide was produced from E. coli transfected with pPGEX-lunasin expression vector and P. pastoris GS115 transfected with pPIC-lunasin expression vector. These recombinant lunasin peptides were similar to the synthetic lunasin peptide in the identification by LC-ESI-MS. In addition, the recombinant lunasin peptide from E. coli and P. pastoris was bound in the chromatin, and inhibited histone acetylation and the activity of histone acetyltransferase. These findings suggest that the production of the lunasin peptide using E. coli and P. pastoris will be useful for industrial utilization of lunasin peptide.

In this study, we produced the recombinant lunasin peptide using E. coli and P. pastoris, and evaluated biological activity of the recombinant lunasin peptide. Lunasin peptide was produced from E. coli transfected with pPGEX-lunasin expression vector and P. pastoris GS115 transfected with pPIC-lunasin expression vector. These recombinant lunasin peptides were similar to the synthetic lunasin peptide in the identification by LC-ESI-MS. In addition, the recombinant lunasin peptide from E. coli and P. pastoris was bound in the chromatin, and inhibited histone acetylation and the activity of histone acetyltransferase. These findings suggest that the production of the lunasin peptide using E. coli and P. pastoris will be useful for industrial utilization of lunasin peptide.

Ornithine deacetylase (argE) gene as a negative selection marker gene was successfully co-transformed with SOD-APX or NDPK2 as a target gene to develop marker free transgenic rice. E.coli argE gene encodes an enzyme which activates N-acetyl phosphinothricin (N-AcPt) through deacetylation. The enzyme was reported that deacetylate N-AcPt, a non cyto-toxic compounds, to produce cyto-toxic PPT. So the argE gene can be used as a negative selection marker and N-AcPt can be used as a substrate of argE gene. Using former purification method (Kriete G., et al., 1996) was very expensive because the small amount of N-AcPt produced by the method. We developed a strategy to produce N-AcPt by means of chemical method. N-AcPt function was identified using hygromycin resistant T1 seeds on MS basal medium contained N-AcPt prior to utilization of argE gene as a negative selection marker in marker free transgenic rice. Negative selection effects were performed with T1 seeds containing argE gene under N-AcPt. This system provides a efficient negative selection effect from transgenic rice and that will be efficiently used for the production of marker gene free rice plants.