• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.439-449
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• 2000

This study was to investigate the expression of transgene after co-injection of spermatozoon and EGFP gene into mature oocytes in cattle. From frozen semen, spermatozoa were treated by DTT with 0.03% Tween-20, freezing and thawing or 0.02% Triton X-100 to disrupt their plasma membranes. The sperm injected oocytes were co-cultured with bovine oviduct epithelial cells in CRlaa, and expression of EGFP in embryos were observbed under epifluorescent microscope. Two pronuclei were formed in oocytes injected with sperm treated by DTT (44.6%), DTT-Tween-20 (48.4%), DTT-freezing (44.4%) and DTT-Triton X-100 (42.9%). Cleavage and blastocyst formation rates of bovine oocytes which injected with sperm treated by DTT, DTT-Tween-20, DTT-freezing, and DTT-Triton X-100 were 49.1, 58.5, 43.9, and 48.4% and 10.2, 13.0, 6.8, and 6.5%, respectively. Although the most of embryos were showing mosaicism, embryos expressing EGFP gene were observed in all treated groups. Therefore, these results indicate that membrane disrupted sperm could interact with exogenous DNA, and that this procedure may be useful to introduce foreign gene into bovine oocytes.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.429-437
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• 2000

This study investigated the successful introduction of genes of erythropoietin (EPO) and enhanced green fluorescent protein (EGFP) in bovine embryos following nuclear transfer of bovine fetal fibroblasts (bFF), which were transfected by retrovirus vector system. Non-starved bFF were, transferred into perivitelline space of enucleated oocytes. The bFF-oocyte units were accomplished by cell to cell fusion and activated with calcium inophore and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CRlaa medium for 8 days. Out of 187 (EPO) and 210 (EGFP) bovine eggs reconstructed by nuclear transfer, 149 (EPO : 80.0%) and 158 (EGFP : 75.2%) embryos were cleaved, and among them 36 (EPO : 24.2%) and 35 (EGFP : 22.2%) embryos developed to the blastocyst stage. Of these blastocysts, 100% integration of EPO gene in 36 embryos was determined by PCR, and 100% expression of EGFP gene in 35 embryos was observed under the fluorescent microscope. This result indicates that bovine oocytes reconstructed by nuclear transfer of transfected bFF can successfully develop to the blastocyst stage. Furthermore, this novel procedure may be presumably an attractive method efficiently to produce the transgenic cattles.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.337-345
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• 1999

The present study was conducted to investigate the effects of various co-culture systems and supplemented protein sources on the in vitro development of bovine IVF embryos. Bovine cumulus oocyte complexes (COCs) were matured and fertilized in vitro. Presumptive zygotes with cumulus cells were transferred to TCM-199 or CRlaa containing 10% FBS or 3mg/$m\ell$ BSA, and cultured for 36~40 hr. After primary culture, cleaved embryos were co-cultured with cumulus cells(CC), bovine oviduct epithelial cells(BOEC) or Buffalo rat liver cells (BRLC) in TCM-199 or CRlaa supplemented with FBS or BSA respectively, for further 6 days. Cleavage rate increased with BSA(P＜0.01) in the both TCM-199(79%) or CRlaa(74%) When embryos were co-cultured with CC or BOEC in TCM-199, blastocyst development was enhanced with BSA(40% and 43%) compared to FBS (22% and 29%) , whereas in CRlaa no difference observed between BSA(40% and 39%) and FBS (40% and 42%). When embryos were co-cultured with BRLC monolayer, FBS enhanced the blastocyst development (P＜0.05) compared to BSA in both TCM-199(41% vs 31%) and CRlaa (44% vs 37%). The result of the present study showed that the cleavage rate of bovine IVF embryos increased with BSA, The result also showed that BSA can enhance the development of IVF embryos in co-culture with CC or BOEC in TCM-199, suggesting the in vitro development is affected by the medium and supplemented protein sources in co-culture with somatic cells.

• 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%).