This study was conducted to investigate the effect of various addition and exclusion time of glucose (Control: no addition, A: 24～72 h, B: 24～48 h, C: 48～72 h, D: 0～72 h, E: 0～48 h, F: 0～24 h and 48～72 h, G: 0～24 h) on embryonic developmental capacity of 2-cell embryos in mice. Developed blastocysts were assessed for mean cell number by differential staining. The zona-intact blastocyst (ZiB) rates were higher (p<0.05) in group B than control. However, the zona-escape blastocyst (ZeB) rates were not significantly different in all groups. At 72 h, total blastocyst (ZiB + ZeB) formation rates were not significantly different in all groups. The mean cell number was not significantly different among all groups. The inner cell mass (ICM) cell number was higher (p<0.05) in group F than control, group A, B and G. The trophectoderm (TE) cell number was higher (p<0.05) in control than group A and D. The %ICM was higher (p<0.05) in group C, D and F than control. The ICM : TE ratio was not significantly different in all groups. Between control and glucose group, no significant difference was observed in the total blastocysts (ZiB + ZeB) formation rates. Also, no significant difference was observed in the mean cell number, ICM cell number and ICM : TE ratio. However the TE cell number was higher (p<0.05) in control than glucose group and %ICM was higher (p<0.05) in glucose group than control. In conclusion, glucose added in culture medium was not inhibitory on blastocyst formation but glucose added for 48 ～72 h in culture medium increases %ICM of blastocysts in mice.

본 연구는 동물복제 및 형질전환 동물생산 등의 연구에 기초자료를 제공하고자 재래산양의 oocyte를 단위발생을 유도하여 회수난자의 조건, 활성화방법, 배양조건 등이 단위발생란의 체외발달율에 미치는 영향을 조사하여 재래산 양의 최적의 배양조건을 확립하고자 실시하였다. 난자의 회수는 체중 15～25 Kg 전 ㆍ 후의 성숙한 미경산 재래산양에 FSH와 PMSG 를 사용하여 과배란을 유기하여 hCG 투여 후 제 35시간째에 외과적인 방법으로 in vivo (체내성숙) 난자는 난관을 관류하는 방법으로 회수하였고, in vitro (체외성숙) 난자는 난포로부터 흡입하여 난포란을 채취하여 약 22시간 체외성숙을 실시하였다. 활성화 처리는 전기자극법과 Ionomycin + 6-DMAP를 처리하여 단위발생을 유도하였다. 복제수정란의 배양은 M16, TCM-199 및 mSOF 배양액으로 6～7일 동안 체외배양을 실시하였다. 활성화를 유도하기 위하여 전기자극 및 ionomycin + 6-DMAP 처리를 하였을 때 분할율은 각각 64.1 및 76.5%로서 이들간에 차이는 없었다. 배반포기로의 발달율은 전기자극방법으로는 전혀 발달이 이루어지지 않았으나, ionomycin +6-DMAP 처리방법에서는 15.6%가 배반포로 발달하였다. In vivo 난자와 in vitro 난자를 활성화를 유도하였을 때 분할율은 86.8 및 69.0%로서 이들간에 유의적인 차이는 없었다. 4-세포기(93.9% vs 66.1%), 8-세포기(90.9% vs 37.0%) 및 상실배기(89.4% vs 23.6%)는 이들간에 유의적(P<0.05)인 차이가 있었으며, 배반포기로의 발달율은 체내성숙난자가 50.0%로서 체외성숙난자의 0.8%보다는 유의적으로 높았다. 활성화를 유도한 난자를 mSOF 배양액으로 체외배양을 실시하였을 때 분할율은 81.0%로서 TCM-199 +oviduct cell 의 64.3% 및 Ml6 배양액의 51.6%보다는 높게 나타났다. 배반포기로의 발달율은 mSOF 배양액에서는 3.4%가 발달하였으나, TCM-199+ oviduct cell 배양액과 M16 배양액에서는 전혀 발달이 이루어지지 않았다. 이상의 결과는 포유동물 난자의 단위발생의 유도 및 체외배양시 난자의 공급원, 난자의 활성화 방법 및 배양조건 등이 차후 단위발생란의 체외발생율에 크게 영향을 미칠 수 있는 근거를 제시해 준다.

본 연구는 재래산양의 핵이식을 실시하여 공여세포의 조건, 전기적 세기 및 융합횟수 등이 융합율과 체외발달율에 미치는 영향을 조사하여 최적의 융합조건을 확립하고자 실시하였다. 공여세포는 귀 유래 섬유아세포와 태아 유래 섬유아세포 2종류를 분리 배양하여 사용하였으며, 수핵란의 채취는 성숙한 미경산 재래산양에 과배란을 유기하여 hCG 투여 후 제 35시간째에 외과적인 방법으로 in vivo (체내성숙)난자는 난관을 관류하는 방법으로 회수하고 in vitro (체외성숙)난자는 난포로부터 흡입하여 난포란을 채취하여 약 22시간 체외성숙을 실시하였다. 수핵난자는 난구세포를 제거한 다음 0.05 M sucrose를 처리하여 세포질이 양호하고 극체가 뚜렷하게 보이는 난자만을 선별하여 핵이식을 실시하였다. 핵이식란의 융합은 전기자극방법으로 융합을 실시하였으며, 핵이식 조작 후 약 3시간 동안 전배양을 실시한 다음 활성화를 유도하였다. 복제수정란은 0.8% BSA가 첨가된 mSOF 배양액으로 6∼7일 동안 체외 배양을 실시하였다. 귀 유래 섬유아세포를 공여세포로 사용하였을 때 융합율은 60.4%로서 태아 유래 섬유아세포의 40.3%보다는 높게 나타났다. 분할율에 있어서는 귀 유래 섬유아세포와 태아 유래 섬유아세포가 각각 47.6 및 48.2%로서 차이가 없었다. 2.40∼2.46 ㎸/㎝로 전기자극을 주었을 때 융합율은 43.8%로서 1.30∼l.40 ㎸/㎝(26.7%)와 2.30∼2.39 ㎸/㎝ (34.8%)가 높게 나타났으며, 융합이 이루어진 핵이식란의 분할율은 82.9(1.30∼l.40 ㎸/㎝), 43.8(2.30∼2.39 ㎸/㎝) 및 51.8%(2.40∼2.46 ㎸/㎝)로서 전기자극의 세기에 따른 유의적(p＜0.05)인 차이는 없었다. 전기융합을 1회 실시하였을 때 in vivo 난자는 43.5%로서 in vitro 난자의 23.6%보다 유의적으로 높게 나타났으며, 2회 실시하였을 때는 55.7(in vivo) 및 39.2%(in vitro)로 in vivo에서 높게 나타났다. 3회 자극을 주어 전체 융합율은 in vivo가 66.1%로서 in vitro의 52.8%보다는 유의적으로 높게 나타났다.

This study was performed to test the cellulose digestibility using the transgenic pigs harboring cellulose degradation gene D (CelD). After delivered offsprings between normal pig and transgenic swine, DNA was isolated from piglets tail for PCR analysis. In first generation, five out of 65 piglets showed CelD positive. Unfortunately, four CelD-positive pigs were died during growing, but one survived pig was used as a transgenic founder to produce F₁ descendents. Among 3 F₁ transgenic pigs produced, one died and the remaining two pigs were used to test the fiber digest efficiency. An assorted feed was composite of 5％ fiber with other ingredients. The feed of 3 kg per day was provided to the pigs including transgenic founders and littermate controls. The manure quantity was measured daily for a month, and all manures were dried for three days to analysis nitrogen, phosphate and fiber concentrations. The fiber digestion efficiencies of the transgenic F₁ pigs showed approximately 10％ higher than those of control pigs. Fiber digestion was not greatly improved in transgenic pigs as it had been expected approximately 30％. Nitrogen concentration of transgenic pig′s manure was slowly decreased compare to the control pigs. Because there were only two transgenic pigs tested, a large number of transgenic pigs may be necessary to obtain more reliable data. Breeding of animals to obtain sufficient transgenic pigs subjected for a further study is on progress. Taken together, this study demonstrated successful production of transgenic pigs with increase of cellulose digestibility in the porcine feed.

The present study were performed to analysis the hematocrit and the red blood cells content into the blood plasma of the transgenic pigs harboring recombinent human erythropoietin gene (rhEPO). Mouse whey acidic protein (mWAP) linked to rhEPO gene was microinjected into pronuclei of porcine one-cell zygotes. After delivered of offspring, PCR analyses identified one mWAP-rhEPO transgenic founder offspring(F/sub 0/). The first generation of transgenic pig (F/sub 0/) harboring mWAP-hEPO appeared to be a male, and the second generation (F₁) pigs were made by natural mating of F/sub 0/ with domestic swine, and male and female transgenic pigs (F₁) were identified by PCR. The blood samples from transgenic and normal pigs were collected for 50 days during lactation and were counted the red blood cell (RBC) numbers and Hematocrit (HCT) content into the blood. The transgenic pigs expressing rhEPO in their blood gave rise to higher RBC numbers and HCT contents than control animals. rhEPO was secreted both in the blood and milk of genetically engineered pigs harboring rhEPO gene. Therefore, this study provides a model regarding the production of transgenic pig carrying hEPO transgene for biomedical research.

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.