• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.135-140
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• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• Development and Reproduction
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• v.19 no.2
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• pp.79-84
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• 2015

The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 ${\mu}M$ roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were $98{\pm}35.2$ and $145{\pm}11.2$, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches.

• Korean Journal of Animal Reproduction
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• v.13 no.2
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• pp.113-120
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• 1989

In order to determine the sex of preimplantation embryos prior to transfer in cattle, a series of experiments were carried out using 45 Holstein donor cows to examine the ovarian response on the gonadotropin and PGF2${\alpha}$, and the morphology of fresh embryos or frozen/thawed embryos after deep freezing at -196$^{\circ}C$. The sexing of embryos treated with the medium containing H-Y antiserum(10%, v/v) and FITC anti-mouse IgG(10%, v/v) were analysed by chromosomal analysis, and the sex of the embryos which survived were ascertain after delivering the pups. The results obtained were summarized as follows ; 1. The average number of developed follicle and corpus luteum per cow were 13.5 and 8.1, and the ovalation rate was 60.1%. 2. Of 220-ova recovered, 75(34.1%) were morula and 91(41.4%) were blastocyst, and the morphological normal and abnormal rate of ova recovered were 75.5% and 24.5%, respectively. 3. Of 39 frozen/thawed embryos, the scores of normal morula and blastocyst, after thawing were 79.2%(19/24) and 73.3%(11/15). The average rate of frozen/thawed embryos which appeared morphologically normal post thawing was 76.9%(30/39). 4. The sex ratio was measured using the embryos treated with immunofluorescence assay to examine the relationship between embryo developmental stage, sex ratio of morula stage embryo was 42.2%(19/45) fluorescing and 57.8%(26/45) non-fluorescing, on the other hand, the ratio switched to 46.8%(29/62) fluorescing and 53.2%(33/62) non-fluorescing embryo in blastocyst stage. The sex ratio was also measured between fresh and frozen/thawed embryos, fresh and frozen/thawed treated embryos were indicated 45.8%(38/83) fluorescing, 54.2%(45/83) non-fluorescing and 41.7%(10/24) fluorescing, 58.3%(14/24) non-fluorescing. This trend indicated the approximal sex ratio was 1 : 1. 5. The result of karyotype test showed the successful rate of sexing embryo is fluorescing and non-fluorescing was 21.2%(7/33) and 29.6%(8/27). The female to male ratio within 33 fluorescing was 28.6 : 71.4, and the ratio of 27 non-fluorescing embryos was 87.7 : 12.5. 6. Of the embryo transferred after assignment of H-Y phenotype, five of the fluorescing embryos survived to term, all was males. Whereas six non-fluorescing embryos also survived to term and the sexes of the calves were 1 male 5 female.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.21-27
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• 2004

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Recipient bovine and porcine oocytes were obtained from slaughterhouse and were matured in vitro according to established protocols. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS and primary fibroblast cultures were established in TCM-199 with 10% FBS. The matured oocytes were dipped in D-PBS plus 10% FBS ＋ 7.5 $\mu$ g/ml cytochalasin B and 0.05M sucrose. Enucleation were accomplished by aspirating the first polar body and partial cytoplasm which containing metaphase II chromosomes using a micropipette with an out diameter of 20∼30 $\mu$m. A Single donor cell was individually transferred into the perivitelline space of each enucleated oocyte. The reconstructed oocytes were electric fusion with 0.3M mannitol fusion medium. After the electrofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. And porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6 ∼8 day at $39^{\circ}C, 5% CO_2 $in air. Interspecies nuclear transfer by recipient bovine oocytes were fused with electric length 1.95 kv/cm and 2.10 kv/cm. There was no significant difference between two electric length in fusion rate(47.7 and 44.6%) and in cleavage rate(41.9 and 54.5%). Using electric length 1.95 kv/cm and 2.10 kv/cm in caprine-porcine NT oocytes, there was also no significant difference between two treatments in fusion rate(51.3 and 46.1%) and in cleavage rate(75.0 and 84.9%). The caprine-bovine NT oocytes fusion rate was lower(P＜0.05) in 1 pulse for 60 $\mu$sec(19.3%), than those from 1 pulse for 30 $\mu$sec(50.8%) and 2 pulse for 30 $\mu$sec(31.0%). The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(53.3%) and 2 pulse for 30 $\mu$sec(50.0%), than in 1 pulse for 60 $\mu$sec(18.2%). The caprine-porcine NT oocytes fusion rate was 48.1% in 1 pulse for 30 $\mu$sec, 45.2% in 2 pulse for 30 $\mu$sec and 48.6% in 1 pulse for 60 $\mu$sec. The cleavage rate was higher(P＜0.05) in 1 pulse for 30 $\mu$sec(78.4%) and 1 pulse for 60 $\mu$sec(79.4%), than in 2 pulse for 30 $\mu$sec(53.6%). In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer and 30.6% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than parthenotes(37.4%).

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.47-52
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• 2016

The objective of this study was to investigate the result of in vivo embryo collection and pregnancy rate after embryo transfer using sex-sorted sperm of Korean brindle cattle. Donor Korean brindle cattle superovulation treated by decreasing dose of FSH injection. Embryos were recovered on 7 days after the third artificial insemination. Control group semen straw used artificial insemination contained 20 million sperm. Sex-sorted semen straws contained 4 million sperm or 10 million sperm. As for the result of the recovery of the in vivo embryos derived from sex-sorted sperm, the number of transferable embryos was significantly highly recovered to be $6.20{\pm}2.28/donor$ from the control group and was significantly lowly recovered to be $1.57{\pm}1.72/donor$ from the group treated at a sperm concentration of $10{\times}10^6$ (p<0.05). The number of unfertilized embryo was $0.8{\pm}1.30/donor$ in control group which was significantly lower than the group treated at a sperm concentration of $4{\times}10^6$ (p<0.05). However, there was no significant difference in the number of undeveloped ova between control and treatment groups. Pregnancy rate after embryo transfer was shown to be 35.00% in control group and 12.50% in treatment group. The karyotype analysis of the calf derived from sex-sorted sperm resulted in a similar chromosomal distribution pattern (2n=60, XX) compared to those of common Korean native cattle.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.163-168
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• 2005

The objective of this study was to produce superior cows by sexual distinction of embryos collected from the donor with pedigree information. Collected embryos distinguished by biopsy using punching or bisection methods and Loop-mediated isothermal amplification of sex-determined DNA for sexing. Six embryos predicted as female were transplanted to recipients and then 2 pregnant cows were normally delivered of calves at 278 and 285 days after embryo transfer. Birth weights of calves named Barani and Borani were 18kg and 25kg, respectively. Adjusted body weights for 90 days were 61.1kg and 88.8kg, respectively. Average daily gains were 0.48kg and 0.71kg, respectively.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.365-373
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• 2000

The purpose of this study was to investigate the effects of the fusion pulses and fusion media on fusion rate and the development of embryos produced by somatic cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus and fetal fibroblast cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 38.5$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. The cumulus cell and cytoplast were fused using one pulse of 70 volts for 40$mutextrm{s}$, two pulses of 70 volts for 40$mutextrm{s}$ and one pulse of 180 volts for 15$mutextrm{s}$. The fetal fibroblast cell and cytoplast were fused using one pulse of 180 volts for 15$mutextrm{s}$ or 30$mutextrm{s}$. The cumulus cell and cytoplast were fused using mannitol and Zimmerman cell fusion medium (ZCFM) as a fusion medium. The fused embryos were activated after the fusion with 10 $\mu$M calcium ionophore for 5 min and 2 mM 6-dimethyl- aminopurine for 3 h. The nuclear transfer embryos were cultured in 500 ${mu}ell$ well of modified CR1aa supplemented with 3 mg/$m\ell$ BSA in th $\varepsilon$ four well dish cove red with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/$m\ell$ BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at 38.5$^{\circ}C$. When the cumulus cells were fused with enucleated oocytes by three different fusion pulses, one pulse of 180 volts for 15 $mutextrm{s}$ yielded the highest fusion rate and developmental rate to blastocyst among the pulses (P＜0.05). When the fetal fibroblast cells were fused with enucleated oocytes, one pulse of 180 volts for 30$mutextrm{s}$ yielded significantly higher fusion rate compared with that for 15 $mutextrm{s}$(P＜0.05). The present result indicates that the fusion rate between karyoplast and cytoplast was affected by the cell type and the optimal fusion condition was different according to cell type or size. When the fusion was conducted by the use of mannitol and ZCFM, the fusion rate was 71.2% and 65.8%, respectively. The developmental rates to blastocyst were 37.8% and 39.8%, respectively. There was no significant difference between two fusion media in the developmental rate of cumulus cell nuclear transfer embryos. These results indicate that optimal electric current should be selected according to cell type.

• Journal of Embryo Transfer
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• v.21 no.3
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• pp.191-198
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• 2006

The objectives of this study was to produce valuable offsprings of Hanwoo and Jeju black cattle using in vivo embryo production and embryo transfer techniques during 5 years ($2001{\sim}2005$) in Jeju. Two hundred and eighty six Hanwoo and sixty nine Jeju black cattles, at random stages of the estrous cycle, received a CIDR. Seven days later, the animals were superovulated with a total of 400 mg pFSH ($Folltropin^{(R)}-V$) administered twice daily in constant doses (each 50 mg) over 4 days. On the 6th administration of FSH, CIDR was withdrawn and 25 mg $PGF_{2{\alpha}}$ was administered. Cows were artificially inseminated thrice after estrous detection at 12 hr intervals. The cows received $250{\mu}g$ GnRH at the time of 2nd insemination. Embryos were recovered 7 or 8 days after the 1st insemination. Recipients were synchronized with donors by insertion of a CIDR for 7 days and administration of 25 mg $PGF_{2{\alpha}}$ at the time of CIDR removal. The collected embryos were transferred to 1,219 recipients by 6 transfer persons. The mean numbers of total ova and transferable embryos from Hanwoo and Jeju black cattle donors were 7.4 and 4.7, respectively The number of transferable embryos differed between Hanwoo (5.0) and Jeju black cattle (3.5, p<0.05), while that of total ova did not differ. Repeated superovulation treatments decreased (p<0.05) the ratio of numbers of the flushed animals vs. superovulated animals in Jeju black cattle, and the numbers of total ova and transferable embryos in Hanwoo. More transferable embryos were collected at summer (5.6) than winter (2.9, p<0.01). The mean pregnancy rate was 40%. The pregnancy.ate was affected by transfer year (2001<2004, p<0.05) and transfer person ($33.0{\sim}41.9%$, p<0.01), while not by donor (embryo) breed. These results showed that in vivo embryo preduction and embryo transfer techniques using CIDR for Hanwoo and Jeju black cattle donors as well as recipient, regardless of their estrous cycle, may enable a stable embryo production and recipient preparation.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.175-180
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• 2006

This study was conducted to examine the effect of pre activation treatment and activation time of recipient cytoplasm on the development of bovine somatic cell nuclear transfer(NT) embryos. Donor cells were transferred and electrofused to enucleated oocytes before(pre-AC) or after activation(post-AC). Activation was induced with a combination of $Ca^{2+}$-ionophore(A23187) and DMAP. NT embryos were cultured in CR1aa containing 3 mg/ml BSA for 9 days. Some NT embryos were fixed at 0.5 to 2.5 hr after fusion(for post-AC) or activation(for pre-AC) for confocal microscopy. Developmental rate to the blastocyst stage was slightly high in the post-AC group(20.6%) compared to that of pre-AC group(15.3%). However, developmental speed of embryos in the pre-AC group was faster than that of embryos in the post-AC group. Development rates to the blastocyst stage were similar among different activation time before fusion(0.5,2 and 4 hr). The result of the present study suggests that development and nuclear morphology are affected f the activation status of the recipient cytoplasm before fusion.