• Korean Journal of Animal Reproduction
• /
• v.22 no.4
• /
• pp.419-424
• /
• 1998

The present study was carried out to examine the efficiency of cloning of transgenic embryos by nuclear transfer(NT) using gene-injected rabbit embryos. The rabbit embryos at pronuclear stage were microinjected with methallothionein-human growth hormone(MT-hGH) gene and cultured to 8- and 16-cell in TCM-199 containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% $CO_2$incubator. The recipient oocytes were collected from the oviducts 14~16 h after hCG injection. The oocytes were enucleated and activated with 5$\mu$M ionomycin and 2mM 6-dimethylaminopurine. Blastomeres form gene-injected embryos were transferred into the enucleated oocytes by micromanipulation. The nuclear transplant oocytes were electrofused and co-cultured with rabbit oviductal cells. Following 120 h of culture, blastocysts were prepared for gene analysis by polymerase chain reaction(PCR). In previous experiment, the rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased while developing to blastocyst(25%)(Kang et al., 1998). The fusion rate of gene-injected blastomeres was significantly(P＜0.05) lower than non-injected blastomeres(66% vs 80%). However, the NT embryos that were derived from gene-injected donor embryos did not differ from control embryos in development to the blastocyst stage(39% vs 31%). Of the 43 NT blastocysts developed from the gene-injected donor embryos, twelve(28%) were positive for the injected DNA. The results indicate that NT with gene-injected embryos can be successfully used for cloning and multiplication of transgenic embryos, furthermore applicable to improvement of transgenic animal production.

• Journal of Embryo Transfer
• /
• v.10 no.1
• /
• pp.73-82
• /
• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• Journal of Embryo Transfer
• /
• v.10 no.2
• /
• pp.105-113
• /
• 1995

This study was conducted to examine the efficiency of enucleation and blastomere isolation from recipient oocytes and donor embryos, respectively and to determine the effect of oocyte age and electric voltage on the fusion rate and in vitro development of the fused oocytes in rabbit nuclear transplantation. Immature oocytes collected from ovarian follicles were matured in vivo for 12 h in TCM-199 containing FCS and hormones and in vivo matured oocytes were collected 17 to 18 h post-HCG. The fresh and frozen donor embryos of 8- to 16-cell stage were collected from the oviduct of superovulated does. The proportion of successfully enucleated oocytes was greatly lower in in vitro matured oocytes (42.3%) than that (62.7%) in in vivo matured oocytes The level of cytochalasin B for in vivo matured oocytes did not affect the efficiency of enuleation, but 7.5 $\mu$g /mL cytochalasin B for in vitro matured oocytes showed a high enucleation rate significantly. The isolation efficiency of a single blastomere nucleus did not differ between 8- and 16-cell stage embryos. The percentage of single blastomeres isolated from 16-cell stage fresh embryos after 0.5% pronase treatment was greatly higher at 16-min treatment (94.4%) than at 8-min(78. 1%) and the blastomeres(61.5%) isolated from frozen-thawed embryos after 16-min pronase were significantly fewer than those of fresh embryos. The age of recipient oocytes affected nuclear fusion rate. The reconstituted oocytes fused at 24-h age showed slightly higher fusion rate (77.8%) than those (65.0%)fused at 18-h age. The fusion rate of in vitro and in vivo matured oocytes inserted with fresh blastomere did not differ among electric voltages, but the cleavage rate and development to morula-blastocysts of in vitro matured oocytes was more higher under 0.6 kV/cm than under 0.8 to 1.2 kV/cm, while the cleavage rate and development of in vivo matured oocytes was higher under 0.8 to 1.0 kV/cm than under 1.2 kV/cm. The fusion and cleavage rate fol1owing insertion with frozen-thawed blastomere was not different between the in vitro and in vivo matured oocytes and was similar to those from fresh blastomere insertion.

• Journal of Veterinary Clinics
• /
• v.12 no.1
• /
• pp.877-886
• /
• 1995

The recycling nuclear transplantation(NT) technique has the powerful potential of producing a large number of genetically identical embryos and offsprings from one embryo. Multiple generational cloning by this technique utilizes the NT embryo itself as the donor for the next generation of cloning. In this experiment, we have produced the third generational cloned embryos by recycling NT. Further we examined comparatively the electrofusion rate and in vitro developmental potential in the cloned embryos of the first second and third generations. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulberco's phosphate buffered saline containing 10 % fetal calf serum(FCS) at 47 hours after hCG injection. In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gl/S transition of 32-cell stage. The first and second generation NT embryos developed to 16-cell were used as donor nuclei for second and third generation. The recipient cytoplasms were utilized the oocytes collected at 14 hours after hCG injection, following revoming the nucleus and the first polar body by micromanipulation. The separated blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were fused by electrical stimulation. The electrofusion rate was seen to be 78.0, 88.0 and 90.3 % in the first second and third generation NT rabbit embryos, respectively. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10 % FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The in vitro developmental potential to blastocyst stage was significantly(P<0.05) decreased in the third(7.2 %) generation NT embryos compared to the first(53.1 %) and second(16.1 %) generation NT embryos. Following in vitro development to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The mean blastomere numbers and cell cycle numbers of NT embryos during the culture period were significantly(p<0.05) decreased in the second(93.9 cells and 6.55 cylces) and third(81.5 cells and 1.35 cylces) generation, compared to the first(189.9 cells and 7.55 cylces) generation.

• The Korean Journal of Nuclear Medicine
• /
• v.38 no.2
• /
• pp.161-170
• /
• 2004

Molecular Nuclear Neuro-Imaging in "CNS" drug discovery and development tan be divided into four categories that are clearly inter-related.(1) Neuroreceptor mapping to examine the involvement of specific neurotransmitter system in CNS diseases, drug occupancy characteristics and perhaps examine mechanisms of action;(2) Structural and spectroscopic imaging to examine morphological changes and their consequences;(3) Metabolic mapping to provide evidence of central activity and "CNS fingerprinting" the neuroanatomy of drug effects;(4) Functional mapping to examing disease-drug interactions. In addition, targeted delivery of therapeutic agents could be achieved by modifying stem cells to release specific drugs at the site of transplantation('stem cell pharmacology'). Future exploitation of stem cell biology, including enhanced release of therapeutic factors through genetic stem cell engineering, might thus constitute promising pharmaceutical approaches to treating diseases of the nervous system. With continued improvements in instrumentation, identification of better imaging probes by innovative chemistry, molecular nuclear neuro-imaging promise to play increasingly important roles in disease diagnosis and therapy.

• Korean Journal of Veterinary Research
• /
• v.33 no.1
• /
• pp.151-169
• /
• 1993

The present study was carried out to investigate the best condition for nuclear-cytoplasm fusion and in vitro culture of nuclear transplanted embryos and to investigate the production of nuclear transplanted offsprings. The nuclei from 2-, 4- and 8-cell mouse embryos were transferred into enucleated 2-cell embryos, and the reconstituted embryos were submitted to direct current(DC) pulses at output voltage of 1.0, 1.5 and 2.0 kV/cm for 100, 150 and $200{\mu}$ sec to induce cell fusion. 1. The culture of intact or zona cut 2-cell embryos in the medium supplemented with cytochalasin B($5{\mu}g/m{\ell}$) and colcemide($0.1{\mu}g/m{\ell}$)for 30 and 60 minutes did not affect the development to later stage. 2. The in vitro developmental rates of group A(a nucleus from one of the blastomeres was removed) and B(electrofusion of group A) were significantly lower than that of control group(p<0.01). 3. When nuclear transplanted embryos were submitted to electrofusion, the significantly higher fusion rates of 2-cell donor nuclei were achieved at the electric field strength of DC 1.5kV/cm for 100 and $150{\mu}$ sec, DC 2.0 kV/cm for $100{\sim}200{\mu}$ sec than DC 1.0 kV/cm for 100 and $150{\mu}$ sec(p<0.01). The significantly higher fusion rates of 4-cell donor nuclei were achieved at DC 2.0 kV/cm for 100 and $150{\mu}$ sec than DC 1.0kV/cm for $100{\sim}200{\mu}$ sec(p<0.01). These fusion rates in 8-cell donor nuclei were 88.7~99.3%. 4. The developmental potency to blastocyst in 2- and 4-cell donor nuclei was significantly higher in DC 1.0 and 2.0 kV/cm for $100{\sim}200{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group (p<0.01). The developmental potency to blastocyst in 8-cell donor nuclei was significantly higher in DC 2.0 kV/cm for $100{\mu}$ sec treated group than in DC 1.0 kV/cm for $100{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group(p<001). 5. The developmental potency to blastocyst after nuclear transplantation was significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 6. The success rate of nuclear injection into enucleated 2-cell embryos was significantly higher in 2-cell donor nuclei than in 4- or 8-cell donor nuclei(p<0.01). 7. The culture time taken for the nuclear transplanted 2-cell embryos to blastocyst stage was significantly longer in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 8. There was no significant difference in the developmental potency of nuclear transplanted embryos within the concentration of EGF at 0 to 15 ng per $m{\ell}$ of BMOC-3 solution. 9. The production rates of offspring after transfer of nuclear transplanted embryos to recipient mouse were significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01).

• The Korean Journal of Nuclear Medicine
• /
• v.35 no.4
• /
• pp.251-257
• /
• 2001

Purpose: We investigated the possibility of early postoperative Tc-99m DTPA scintigraphy in predicting long-term renal transplant survival. Materials and Methods: 64 living donor (LD) grafts were divided into two groups according to the graft function on early post-operative renal scintigraphy. Survival analysis was performed using Kaplan-Meier method and Cox proportional hazard model. Chi-square test was performed to evaluate the difference in the frequency of acute rejection. Results: Cumulative renal transplant survival was decreased in 11 patients with abnormal renal scintigraphy, but it was not statistically significant. Decreased graft function on early post-operative renal scintigraphy was not a predictor of long-term graft survival. The frequency of acute rejection was higher in abnormal scintigraphy group, and the difference was statistically significant. Conclusion: Decreased graft function on early post-operative renal scintigraphy has no direct effect on long-term renal transplant survival in LD transplantation. But it may have an indirect elect through increasing the frequency of acute rejection.

• Journal of Embryo Transfer
• /
• v.12 no.3
• /
• pp.259-267
• /
• 1997

To improve the efficiency of production of cloned embryos and animals by nuclear transplantation in the rabbit, the effect of cell cycle of donor nuclei and type of recipient cytoplasm on the in vitro developmental potential and production efficiency of offspring was determined. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G$_1$ phase of 32-cell stage. The oocytes collected at 14h post-hCG injection were freed from cumulus cells and then enucleated. One group of the enucleated cytoplasms was activated by electrical stimulation prior to injection of donor nucleus, and the other group was not pre-activated. The separated G$_1$phase blastomeres of 32-cell stage embryos were injected into the perivitelline space of recipient cytoplasms. After culture for 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation and the fused nuclear transplant embryos were co-cultured for 120h and the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. Some of the nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The electrofusion rate was similar between the types of donor nuclei and recipient cytoplasms used. However, the nuclear transplant embryos using G$_1$ phase donor nuclei were developed to blastocyst at higher rate(60.3%) than those using S phase ones(24.7%). Also, when non-preactivated oocytes were used as recipient cytplasms, the develop-mental rates of nuclear transplant embryos to blastocysts were significantly(P< 0.05) higher(57.1%) than those using preactivated ones(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased signficantly(P<0.05) more in the non-preactivated recipient cytoplasm(163.7 cells), as compared whit the preactivated recipient cytoplasm(85.4 cells), A total of 49 nuclear transplant embryos were tranferrid into 5 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer. these results showed that the blastomeres of G1 phase and non-preactivated oocytes might be utillzed efficiently as donor nuclei and recipient cytoplasms in the nuclear transplant procedure, thought the offspring production remained still low.

• Journal of Embryo Transfer
• /
• v.8 no.1
• /
• pp.9-12
• /
• 1993

포유동물의 초기 발생단계에서 핵의 분화와 전능성을 규명하고 제2세대 핵이시 기법을 개발하고자 생쥐를 모델로 하여 공핵란은 2-세포기에 있는 수정란의 핵을 사용하였으며, 수핵란은 zygote 및 2-세포기에 있는 수정란을 탈핵하여 제2세대 핵이식을 실시하여 electrofusion system으로 핵융합을 실시하고 cloned embryo를 작출하여 이를 24-48시간동안 체외에서 배양을 시킨 다음 위임신이 유기된 수란생쥐의 난관에 체내 이식을 실시하여 개체로의 발생 여부 등을 조사하였다. 핵이식후의 융합율은 zygote 및 2-세포기의 수정란을 수핵란으로 사용하였을 때 각각 84.7 및 84.0%으로서 차이가 없었으며, 제1세대의 86.8 마ㅊ 85.4%로서 세대간에 차이가 없었다. 4-세포기 이상으로 발달한 제2세대 핵이식 수정란의 체외배양율은 수핵란을 zygote 및 2-세포기 수정란을 사용하였을때 각각 36.2 및 43.7%로서 제1세대 핵이식의 44.3 및 50.4% 보다는 다소 낮았다. 제2세대 핵이식 수정라늘 위임신이 유기된 수란생쥐의 난관에 이식을 실시하여 얻은 산자생산율은 수핵란을 zygote 및 2-세포기 수정란을 사용하였을때 각각 23.0 및 25.0%로서 모두 25마리의 산자를 생산하였다.

• The Korean Journal of Nuclear Medicine
• /
• v.25 no.1
• /
• pp.95-100
• /
• 1991

Computerized scintigraphv using $^{99m}Tc-DTPA$ was performed to 37 isolated rabbit kidneys after preservation for 48 hours in perfusates differing in their compositions, i. e., Group 1 (N 9) in Collins' solution, Group 2 (N 10) in Collins' plus trifluoperazine, Group 3 (N 9) in Collins' plus urokinase and Group 4 (N 9) in Collins pius urokinase plus verapamil. Satisfactory images, and statistically analyzable quantitative indices such as perfusion score, filtration rate and cortical uptake ratio (CUR) were obtained by the evaluations of first-pass perfusion, equilibration slopes and postequilibration images. Significant improvements in CUR were observed by adding trifluoperazine (Group 2) and urokinase (group 3) as compared to Collins' only group (Group 1), p<0.05 for each, and all of the three indices (perfusion score, filtration rate and CUR) were also significantly (p=0.0092 p<0.05 and p<0.05) improved by adding urokinase plus verapamil (Group 4). We concluded that the computerized scintigraphy with Tc-99m DTPA provide valuable quantitative indices for evaluation of preserved kidney funcitions and suggest its possible clinical applicability in cadaver kidney transplantation considering the safety and easiness of the prodedure.