This study was carried out to examine on developmental competence of Hanwoo embryos cultured in vitro according to culture conditions and freezing methods. The in vitro developmental competence to blastocyst stage at Day 8 of culture in SOF was significantly (p<0.05) higher than that in CR1aa (30.3% vs. 18.4%). The in vitro developmental rate of morula and blastocysts cultured in group culture was significantly (p<0.05) higher than that in individual culture (41.4% and 36.0% vs. 21.1% and 10.5%, respectively). The cell number of Day 8 blastocysts in group culture was significantly (p<0.05) higher than that in the individual culture (, respectively). The survival rates of frozen-thawed balstocysts that were exposed in 1.5 M ethylene glycol or 1.5 M ethylene glycol containing 0.1 M sucrose were 77.5% and 78.7%, respectively. The survival rates of blastocysts cultured for 48 h in slow freezing and vitrification was not significantly different (73.3 and 74.0%). In conclusion, in vitro developmental competence of bovine embryos was influenced on the culture medium (SOF) and culture method (Group culture). Survival rate of frozen-thawed of bovine embryos was not influenced on freezing solutions and freezing methods.

Our goal was to examine the effects of early denudation on the enucleation efficiency and developmental competence of embryos following somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA). Oocytes were denuded following 30 h of in vitro maturation (IVM) and then cultured with (D+) or without (D-) their detached cumulus cells for additional h. Control oocytes were denuded after h of IVM. The size of the perivitelline space was larger at 40 h of IVM () than at 30 h ( p<0.01). The distances between the metaphase II (M II) plates and the polar bodies (PBs) were shorter in D+ () and D- oocytes () than in control oocytes ( p<0.01). Enucleation rates following blind aspiration at 40 h of IVM were higher (p<0.01) in D+ (92%) and D- oocytes (93%) compared to controls (82%). Early denudation did not affect oocyte maturation or the in vitro development of SCNT and PA embryos. When SCNT embryos from D+ oocytes were transferred to four gilts, pregnancy was established in two pigs, and one of them farrowed three live piglets. In conclusion, early denudation of oocytes at 30 h of IVM could improve the enucleation efficiency by maintaining the M II plate and the PB within close proximity and support the in vivo development of SCNT embryos to term.

This study was carried out to investigate the effect of morphology of oocytes, kinds of media, cysteine and myo-inositol supplementation on IVM rate of porcine oocytes. Cumulus- enclosed oocytes were incubated in maturation NCSU-23 and TCM-199 medium with supplementation with 3, 5, 10, 20 mM myo-inositol and 0.05, 0.1, 0.5, 1.0 mM cysteine. 1. When classified by morphology, excellent, good and fair of cumulus-enclosed oocytes were incubated for 48 hrs and the IVM rate were , respectively. The rate were greater in oocytes with excellent cumulus cells than those without cumulus cells. 2. The IVM rate of oocytes cultured in TCM-199 and NCSU- 23 medium supplementation or non-supplementation with 1.0 mM myo-inositol were , respectively. Supplementation with myo-inositol significantly increased the IVM rate of oocytes. 3. The IVM rate of oocytes cultured in NCSU-23 medium supplementation of 3, 5, 10, 20 mM myo-inositol for 48 hrs were , respectively. The IVM rate of oocytes in NCSU-23 medium supplemented with 10 mM myo-inositol were significantly increased compared to control (). 4. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 media supplement with 0.3, 0.5, 1.0, 2.0 mM myo-inositol were , respectively. The IVM rate of oocytes in NCSU-23 medium supplemented with 10 mM cysteine were significantly increased compared to control ().

This study was performed to investigate the effect of VEGF on in vitro maturation of porcine oocytes. The base medium for IVM, TCM-199 was supplemented with 0.6 mM cysteine, 0.91 mM pyruvate, 10 ng/ml epidermal growth factor, kenamycin, insulin and 10% (V/V) porcine follicular fluid (pFF) as a Group A; Group B was consists of Group A plus 5 ng/ml VEGF; Group C was consists of replacement of pFF by 10% PVA and Group D: was consists of Group C plus 5 ng/ml VEGF. 1. The maturation rate was significantly higher (p<0.05) in control and VEGF+pFF group than other two groups (, respectively). 2. Addition of VEGF without pFF showed a negative effect on oocytes maturation and about 58.26% oocytes were reached to M-II stage. 3. In the parthenogenetic development, the cleavage rate was significantly higher (p<0.05) in control and VEGF+pFF group (, respectively) than other groups (, respectively). 4. The blastocyst formation rate was significantly higher (p<0.05) in VEGF+pFF group () compared to control and other groups. 5. There was no significant difference in cell numbers (inner cell mass or trophectoderm) among these groups.

본 실험은 알코올 발효사료 처리에 따른 in vitro 발효 pattern의 변화를 검토하기 위하여 반추위내 ammonia, pH, alcohol 및 volatile fatty acids 농도와 NDF 분해율에 미치는 영향을 구명하고자 실시하였다. 비지박 알코올 발효사료(AFS)는 비지박(DM 20%)과 시판중인 배합사료(DM 87%)를 각각 50:50의 비율로 혼합하고 당밀 5%, yeast 0.5%를 첨가한 후 에서 24시간 혐기적으로 배양하여 제조

This study was conducted to examine the protein kinase inhibitors, 6-dimethylaminopurine (DMAP) and cycloheximide (CHXM) on the development and chromosome constitution of porcine parthenogenetic embryos. In vitro matured oocytes were activated by electric stimuli (ES) or a combination of ES with culture in 2 mM DMAP or 10 μg/ml CHXM for 4 hr. Activated oocytes were cultured in PZM-3 for 6 days. Some 1-cell embryos and blastocysts were fixed by air dry method to analyze the chromosome constitutions and/or total cell number. Blastocyst development of DMAP-treated group (26.7%) was significantly higher (p<0.05) than those of CHXM-treated and ES control groups. Ploidy in 1-cell stage embryos was not different among groups (77.3 to 81.0%), however, proportion of diploid chromosome constitutions was high in DMAP-treated group (61.9%, p<0.05). In the blastocyst stage, proportion of diploid chromosome plates was significantly high in DMAP-treated group (64.2%, p<0.05), and proportion of abnormal chromosome plates was higher in CHXM-treated group (36.6%, p<0.05) than DMAP-treated group (28.3%,). Proportion of embryos with abnormal chromosome constitutions was slightly increased by DMAP (40.0%) and CHXM (42.1%) treatment due to the increasing of mixoploid (47.4 and 52.0%). The present study shows that the DMAP treatment increase the development of porcine parthenotes. However, parthenogenetic activation by ES or combined treatment with ES and DMAP or CHXM detrimentally affects the chromosome constitutions of porcine parthenotes during early embryonic development, leads to increased abnormal ploidy in the blastocyst stage.

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the capacity to differentiate into various types of cells in the body. Hence, these cells may potentially be an indefinite source of cells for cell therapy in various degenerative diseases including neuronal disorders. For clinical applications of human ES cells, directed differentiation of these cells would be necessary. The objective of this study is to develop the culture condition for the expansion of neural precursor cells derived from human ES cells. Human ES cells were able to differentiate into neural precursor cells upon a stepwise culture condition. Neural precursor cells were propagated up to 5000-fold in cell numbers over 12-week period of culture and evaluated for their characteristics. Expressions of sox1 and pax6 transcripts were dramatically up-regulated along the differentiation stages by RT-PCR analysis. In contrast, expressions of oct4 and nanog transcripts were completely disappeared in neural precursor cells. Expressions of nestin, pax6 and sox1 were also confirmed in neural precursor cells by immunocytochemical analysis. Upon differentiation, the expanded neural precursor cells differentiated into neurons, astrocytes, and oligodendrocytes. In immunocytochemical analysis, expressions of type III β-tubulin and MAP2ab were observed. Presence of astrocytes and oligodendrocytes were also confirmed by expressions of GFAP and O4, respectively. Results of this study demonstrate the feasibility of long-term expansion of human ES cell-derived neural precursor cells in vitro, which can be a potential source of the cells for the treatment of neurodegenerative disorders.

Insulin, transferrin and selenium (ITS) complex is reported to improve in vitro development of oocytes and embryos. This study was carried out to investigate the effects of ITS during in vitro culture (IVC) of porcine parthenogenetic and nuclear transfer (NT) embryos on subsequent developmental capacity in vitro. The electrically activated oocytes were cultured in Porcine Zygote Medium (PZM-3) with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 7 days. Also, the electrically activated reconstructed embryos were cultured in PZM-3 with various concentrations (0, 0.1, 0.5, and 1.0%) of ITS for 6 days. Addition of ITS to culture medium did not affect development of porcine parthenogenetic embryos in vitro. To test the effect of ITS on the in vitro development of porcine NT embryos, factorial experiments were also performed for in vitro maturation (IVM) medium (TCM-199) with or without 1% ITS and culture medium (PZM-3) with or without 0.5% ITS. Addition of 0.5% ITS to culture medium increased (p<0.05) the proportion of NT blastocysts compared with non-treated group. In contrast, addition of 1% ITS to culture medium was ineffective or had a detrimental effect. Also, addition of ITS only to maturation medium increased (p<0.05) the percentage of NT blastocysts formation compared with the control group. In conclusion, addition of ITS to IVM or IVC medium could improve subsequent blastocyst development of porcine NT embryos.

Epigenetic modification dependent DNA methyltransferases (DNMTs) play an important role in tissue- and stage-specific gene regulation and normal mammalian development. In this study, we show that DNMTs are expressed at different levels during hematopoietic stem cell (HSC) differentiation to proerythrocytes. DNMT1, DNMT3A, and DNMT3B were highly expressed at day 7 after differentiation. We used specific siRNA as a tool to probe the relationship between the expression of DNMTs and erythropoietic differentiation. When introduced siRNA of DMNT1 and DMNT3b in human CD34+ cells, these more differentiated into erythrocytes. This was confirmed by glycophorin A (GPA) positive cell analysis and globin gene expression. GPA+ cells increased up to 20～30%, and γ- and ε-globin genes increased in siRNA transfected cells. Therefore, our data suggest that suppression of DNA methylation can affect positively differentiation of HSC and may contribute to expression of erythrocyte lineage genes including GPA and globins.

우리나라 자연 상태의 노지재배에서 종자번식이 되 지 않는 크로코스미아의 기내증식을 위하여 2,4-D, NAA, kinetin, BA 처리가 캘러스 및 소자구 형성 등 에 미치는 영향을 조사하였다. 어린자방 배양에서 캘러 스 형성율은 BA와 2,4-D 혼합 처리에서 1~28%로 나타났으며 기내 뿌리 발생은 2,4-D, NAA 단독처리 에서 양호하였다. 소화를 이용한 배양에서는 kinetin +2,4-D, kinetin+NAA 조합에서 뿌리만 형성되기도 하 였다. rhizome 배양에서 줄기 형성에는 kinetin 단독 처리, kinetin+2,4-D 혼합처리가 양호하였으며 캘러스 형성에는 2,4-D 단독처리, 2,4-D+BA, 2,4-D+kinetin 혼합처리에서 25~92.8%의 형성율을 나타냈으며 corm 형성에서는 kinetin 처리와 kinetin + 2,4-D 0.1 mg·L-1 처리구에서 30~70%의 높은 형성율을 보였다.

This study was conducted to investigate the effect of periods of sperm preincubation, concentrations and storage periods of miniature pig sperm on in vitro penetration of porcine follicular oocytes. High concentration (1×105, 2.5×105, 5×105, 1×106, 5×106 and 1×107) did support sperm penetration than low concentrations (P<0.05). However, polyspermic oocyte rates were increased with high concentrations of sperm. On the other hand, sperm preincubated during 1, 2 or 5h could be penetrated than sperm preincubated during 0, 3 or 4h (P<0.05). When sperm were storaged with different periods, in vitro pentration rates were significantly higher 0～2 days than 3～4 days of sperm storage (p<0.05). These results indicate that sperm treatment factors can effect in vitro penetration in miniature pig.

PDT is an established cancer treatment modality, This can be attri buted to the attractive basic concept of PDT; the combination 0 1' two therapeutic agents , a photosensiti zing drug and light, which are relatively harmless by themselves but combined ultimately cause more 0 1' less selective tumor destruction, The bacteri ochlorophyll - derivatived photosensitizer s are known to be s tabl e and highly effïcient, ln thí s study, we conducted a seríes of experiments to develope the light induced anticancer drugs against oral cancer cell , We tested the cytotoxi city of the hydroxybacteriochlorine by MTT assay and observed the cell death pattern(apoptosis or necrosis) after PDT by hoechst 33342 and propidium iodide s taining methods, lC50 value of the hydroxybac teriochlorine was 3 1 ， 3 ng/ n띠， At higher doses of hydroxybacteriochlori ne () 60ng/ rnQ) , cancer cells died exclus ively by necrosis after PDT By contrast, at lC50 value, hydroxybacteri ochlorine induced cancer cell to undergo apop totic c e ll death, The induct ion begins approximately 6 hours a fter PDT, We inves tigates intrace l1 ular localization of hydroxybacte riochlorine by oral cancel‘ cell via confocal laser scanning mi croscopy, Oral cancer cells dua l-stained with hydrox ybacteriochlo1' ine and organelle-specific flu orescence probes (Mi totracker , Lysotracke1', ER- Tracker) revealed an intracellular f1 uores c ence dis tribution restricted to cyt oplasmic compartments with no det ectable fluorescence in the nucleus, Confocal im ages of cells containing hydroxybacte1'iochl orine were never overla p to mitochondria, lysosome . endoplasmic reticulum when digita lly overla pped with tqe organel1e-specific f1 uorescence p1'obe images o{' the same cells These results demons trated tha t the hydroxybacte1' iochlorine may have a fun ction as a photosensitizer and cytotoxicity hydroxy bacteriochlo1'ine for o1'al cancer cell is more sensitive than head & neck cancer cell 0 1' ce1'vical cancer cell Therefore PDT using hydroxybacte1'iochl orine is suita ble treatment for oral cavity carcinoma patients